KASP molecular marker for identifying high temperature resistance of procambarus clarkii and application thereof

Abstract

The invention belongs to the field of molecular marker screening of aquatic animals, and provides a KASP molecular marker for identifying high temperature resistance of procambarus clarkii and application of the KASP molecular marker. According to the procambarus clarkia high-temperature-resistant gene Hsp70-1 and the procambarus clarkia high-temperature-resistant gene Hsp70-2 disclosed by the invention, two SNP (Single Nucleotide Polymorphism) sites of the procambarus clarkia high-temperature-resistant gene Hsp70-1 and the procambarus clarkia high-temperature-resistant gene Hsp70-2 are remarkably related to high-temperature resistance, Hsp70-1-Hap1 haplotypes and Hsp70-2-Hap1 haplotypes are high-temperature-resistant dominant haplotypes, and the application potential of high-temperature-resistant application through combination of the double genes of KSAP markers developed aiming at the loci are good in typing effect, high in specificity, good in stability, convenient to detect, economical and efficient, and can be widely applied to breeding of high-temperature-resistant varieties of the procambarus clarkii. The KASP marker disclosed by the invention can screen out the procambarus clarkii with the SNP haplotype combination with the high-temperature-resistant advantage, and meets the technical requirements of high-temperature-resistant molecule-assisted breeding of the procambarus clarkii.

Description

Technical Field

[0001] The present invention belongs to the field of screening molecular markers for aquatic animals; specifically, it relates to a KASP molecular marker for identifying high-temperature resistance of Procambarus clarkii and its application. Applying the present invention can improve the high-temperature resistance ability of Procambarus clarkii seedlings, which is of great significance for the breeding of high-temperature resistant varieties (lines) of Procambarus clarkii. Background Art

[0002] Procambarus clarkii is commonly known as crayfish, also known as red swamp crayfish, belonging to the phylum Arthropoda, class Crustacea, order Decapoda, family Cambaridae, genus Procambarus, and is a crustacean living in fresh water.

[0003] The integrated rice-crayfish farming model utilizes the paddy field ecosystem and organically combines rice planting and Procambarus clarkii farming through artificial measures. It has significant advantages such as low production cost and environmental friendliness, and has become the main form of Procambarus clarkii farming in China (accounting for more than 80%). Existing research shows that the integrated rice-crayfish farming model faces significant environmental challenges during the high-temperature season. During the high-temperature period in summer, the average daytime temperature of the paddy field water body can reach above 31°C, and the extreme instantaneous water temperature record value even exceeds 38°C. This heat stress environment directly threatens the survival status of Procambarus clarkii, and its shallow water habitat characteristics exacerbate its temperature sensitivity. The heat stress problem in the industrial chain presents multi-dimensional characteristics: in the logistics and transportation link, when high-temperature weather occurs during the concentrated listing period, the heat stress reaction during transportation can cause significant mortality; at the germplasm resource level, long-term reverse selection and inbreeding have led to the degradation of the germplasm of Procambarus clarkii in China, specifically manifested as genetic disadvantages such as decreased growth performance and attenuated stress resistance. In response to this industrial bottleneck, it is urgent to construct a breeding strategy for stress-resistant new varieties (lines). Through molecular marker-assisted selection, a new strain of Procambarus clarkii with heat tolerance traits is selectively bred. Summary of the Invention

[0004] In view of the many problems existing in the prior art, the purpose of the present invention is to overcome the difficulties in the high-temperature resistance breeding of Procambarus clarkii, and a KASP molecular marker for identifying high-temperature resistance of Procambarus clarkii and its application are established by using known high-temperature resistance genes and KASP genotyping technology.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] The application provides a KASP molecular marker for identifying high-temperature-resistant Procambarus clarkii, relates to two reporter genes, namely, Hsp70-1 (Gene ID: 123774884) and Hsp70-2 (Gene ID: 123775010) for high-temperature-resistant traits of the Procambarus clarkii, and site information is as follows:

[0007]

[0008] The application also provides a KASP molecular marker primer combination for improving the high-temperature-resistant capacity of the Procambarus clarkii, and primer sequence information is as follows:

[0009]

[0010] The application also provides an application of the KASP molecular marker primer combination for improving the high-temperature-resistant capacity of the Procambarus clarkii, and the application comprises the following steps:

[0011] (1) Obtaining of a high-temperature-resistant extreme phenotype population: stress culture of randomly grouped Procambarus clarkii for 108 hours through high temperature (35 DEG C), and counting of death time of individuals in the population. According to the death time, the population is divided into three phenotypes, namely, Sensitive, Tolerant and Strong Tolerant. The individuals dying in 0-36 hours are of the Sensitive phenotype, the individuals dying in 36-72 hours are of the Tolerant phenotype, and the individuals dying in 72-108 hours are of the Strong Tolerant phenotype. At least three high-temperature-resistant extreme phenotype populations are obtained.

[0012] (2) RNA extraction of all Procambarus clarkii individuals obtained in step (1).

[0013] (3) Obtaining of cDNA corresponding to the RNA obtained in step (2) through a reverse transcription kit, and storage at -20 DEG C for standby.

[0014] (4) Design of PCR primers according to NCBI nucleic acid database information, and primer sequence is shown in Table 1.

[0015] Table 1 primer sequence design of Hsp70-1 and Hsp70-2 coding regions

[0016]

[0017]

[0018] (5) PCR amplification of the cDNA sample of the high-temperature-resistant extreme phenotype (sensitive or strong resistant) individual of the Procambarus clarkii obtained in step (1). The specific steps are as follows: amplification of the target fragment by using the primers designed in Table 1, and detection of the integrity of the fragment by agarose gel electrophoresis. The site information of the amplified target fragment is shown in Table 2.

[0019] Table 2 Information of effective SNP sites in coding region of Hsp70-1 and Hsp70-2

[0020]

[0021] (6) According to the sequence information of NCBI nucleic acid database data, KASP molecular markers were developed for SNP sites, and KASP marker primers were designed using Primer 5 software. FAM and HEX fluorescent labels were added to the 5' end of the F1 and F2 forward primers of the two genotypes, respectively. The underlined sequence is the universal fluorescent label. The primer can be designed using the complementary strand sequence of the genome, so that the 3' end base of the F1 and F2 forward primers corresponds to the two alleles before and after mutation, respectively. The sequence information of the KASP marker primers is shown in Table 3.

[0022] Table 3 Sequence of Hsp70-1 and Hsp70-2-KASP marker primers

[0023]

[0024] (7) Extract DNA from all muscle samples of Procambarus clarkii, dilute the sample DNA concentration to 5 ng / μL, and store at -20℃ for subsequent genotyping.

[0025] (8) Genotype at least 3 populations with extreme high temperature resistance phenotype obtained in step (1) using KASP markers. KASP system: 1.4 μL DNA, 0.075 μL of primers F1 and F2, 0.2 μL of primer R, 2.5 μL of KASP-Mix, and 0.75 μL of ddH2O. KASP reaction program: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 sec, 68℃ annealing for 1 min, 10 cycles, each cycle with annealing temperature decreasing by 0.8℃; 95℃ denaturation for 20 sec, 57℃ extension for 1 min, 34 cycles. After the reaction, use LGC high-throughput genotyping instrument for detection, and sequencing to verify the accuracy of genotyping.

[0026] Table 4 Important SNP haplotype combinations of Hsp70-1 and Hsp70-2

[0027]

[0028] (9) Two SNP loci of Hsp70-1 and Hsp70-2 were analyzed using a dual-gene combination to screen for highly resistant genotypes. This invention also provides high-temperature resistant haplotypes of Procambarus clarkii Hsp70-1-Hap1 and Hsp70-2-Hap1; and high-temperature resistant dual-gene combination of Procambarus clarkii Hsp70-1 and Hsp70-2.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] Under high-temperature stress, the red swamp crayfish (Procambarus clarkii) experiences problems such as decreased growth rate, reduced immunity, difficulty in harvesting, and transportation losses, impacting economic benefits. This invention utilizes the SNP differences in heat-resistance genes and employs the KASP method to rapidly screen for SNP combinations with excellent heat-resistance capabilities. Two SNP loci, Hsp70-1 and Hsp70-2, identified in the red swamp crayfish heat-resistance genes, are significantly correlated with heat resistance. The Hsp70-1-Hap1 and Hsp70-2-Hap1 haplotypes are dominant heat-resistance haplotypes, and the combined application of the Hsp70-1 and Hsp70-2 dual genes shows great potential for heat resistance. The KSAP markers developed for these loci exhibit good genotyping effects, high specificity, good stability, convenient detection, and cost-effectiveness, and can be widely applied to the breeding of heat-resistant red swamp crayfish varieties. Furthermore, they can be used as an auxiliary selection tool in heat-resistant breeding of red swamp crayfish.

[0031] (1) The SNP genotypes screened in this invention have excellent heat resistance and stable effects.

[0032] (2) The present invention has high repeatability and wide applicability.

[0033] (3) The present invention can use DNA samples to complete the screening, and has low requirements for sample quality. Attached Figure Description

[0034] Figure 1 The number of individuals from three groups of red swamp crayfish at different time points under 35℃ stress;

[0035] Among them, 0-36h: Sensitive phenotype; 36-72h: Tolerant phenotype; 72-108h: Strong Tolerant phenotype;

[0036] Figure 2 Analysis of the heat resistance of Hsp70-1 and Hsp70-2 gene SNP haplotypes;

[0037] In this diagram, A represents the heat resistance analysis of the Hsp70-1 gene SNP haplotype in three populations; B represents the heat resistance analysis of the Hsp70-2 gene SNP haplotype in three populations. S: Sensitive, T: Tolerant, ST: Strong Tolerant.

[0038] Figure 3 Comparative analysis of dominant and inferior haplotypes of Hsp70-1 and Hsp70-2 genes;

[0039] Among them, GA+CC: is the dominant haplotype Hsp70-1-Hap1+Hsp70-2-Hap1; AA+CT: is the inferior haplotype Hsp70-1-Hap2+Hsp70-2-Hap2.

[0040] Figure 4 Hsp70 gene KASP marker SNP typing diagram;

[0041] in, Figure 4 Figure A in the diagram: Hsp70-1-KASP classification diagram; Figure 4 Figure B in the diagram: Hsp70-2-KASP genotyping diagram. Bottom right cluster: homozygous genotype A; top left cluster: homozygous genotype B; middle cluster: heterozygous genotype H. Detailed Implementation

[0042] The technical solution of the present invention will be further described below through specific embodiments.

[0043] Example 1:

[0044] SNP site mining and heat resistance analysis of the Hsp70-1 gene:

[0045] (1) Obtaining a population with extreme high-temperature resistance phenotypes, the specific method is as follows:

[0046] This experiment constructed three randomized groups, each with a sample size of no less than 150 individuals. The individuals were subjected to high-temperature (35℃) stress culture of *Procambarus clarkii* for 108 hours, and the survival time of each individual within each group was recorded. Based on the time of death, each individual within the group was classified into three phenotypes: Sensitive, Tolerant, and Strong Tolerant. Individuals that died within 0-36 hours were classified as Sensitive, those that died within 36-72 hours as Tolerant, and those that died within 72-108 hours as Strong Tolerant. The three phenotypes were replicated for each labeled individual, resulting in three extreme high-temperature resistant phenotype populations: Population 1 - Hsp70-1, Population 2 - Hsp70-1, and Population 3 - Hsp70-1.

[0047] (2) Extraction of hemolymph RNA from Procambarus clarkii, the specific steps are as follows:

[0048] 500 μL of hemolymph sample was extracted from the red swamp crayfish using a 1 mL syringe and slowly injected into an enzyme-free EP tube. The hemolymph and anticoagulant were gently mixed in equal volumes and placed on ice. The sample was centrifuged at 800 rf for 20 min at 4°C to separate the blood cells. The supernatant was discarded, and the white precipitate was retained. 200 μL of pre-chilled Trizol reagent was added, and the sample was ground evenly using a hand grinder. 800 μL of Trizol reagent was added again, and the sample was allowed to stand at room temperature for 5 min. Then, the sample was centrifuged at 12000 r / min for 10 min at 4°C. 900 μL of the supernatant was transferred to a new EP tube, and 200 μL of chloroform was added. The mixture was thoroughly shaken and allowed to stand at room temperature for 5 min. The sample was then centrifuged at 1200 r / min for 10 min at 4°C. Centrifuge at 0 rpm for 10 min; after centrifugation, the solution will show three layers. Aspirate 400 μL of the supernatant; add an equal volume of 400 μL of isopropanol, mix gently, and let stand at room temperature for 5 min; centrifuge at 12000 rpm for 15 min at 4 °C; discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water, and resuspend the precipitate; centrifuge at 8000 rpm for 5 min at 4 °C; discard the supernatant, aspirate the residual solution, place the EP tube in a fume hood for 5 min to dry thoroughly, and add 20 μL of DEPC water to dissolve the RNA. RNA concentration was determined using a NanoDrop 2000, and RNA extraction quality was confirmed by 1.5% agarose gel electrophoresis.

[0049] (3) cDNA was obtained by reverse transcription using a reverse transcription kit. The specific steps are as follows:

[0050] Mix 8 μL of RNA solution, 2 μL of 5×FastKing-RT SuperMix, and 10 μL of ddH2O in a PCR tube, vortex, and briefly centrifuge. Run the PCR program at 42°C for 15 min and then at 95°C for 3 min. Store the cDNA at -20°C for later use.

[0051] (4) Primers for the Hsp70-1 coding region were designed using Primer5 software based on data from the NCBI nucleic acid database. The primer sequence information for the Hsp70-1 coding region is shown in Table 5.

[0052] Table 5. Primer sequence information for the Hsp70-1 coding region.

[0053]

[0054] (5) The Hsp70-1 coding region fragment was amplified using the above primers. The PCR system consisted of: 10 μL 2×PCR Mix, 1 μL each of F and R primers, 1 μL cDNA, and 7 μL ddH2O. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, for 35 cycles; 72℃ extension for 10 min. The size and quality of the PCR products were detected by 1.5% agarose gel electrophoresis. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared using Sequencher software, which determined that there was a G>A synonymous mutation in the Hsp70-1 coding region of the thermoresistant gene at position NC_091159.1-53803001 in the chromosome.

[0055] (6) Based on the sequence information of the NCBI nucleic acid database, KASP molecular markers were developed for SNP sites. Primers were designed using Primer 5 software, and FAM and HEX fluorescent tags were added to the 5' ends of the F1 and F2 forward primers for the two genotypes, respectively. The underlined part of the sequence is a universal fluorescent tag. KASP marker information is shown in Table 3.

[0056] (7) Extract DNA from the muscle sample of Procambarus clarkii. The specific method is as follows:

[0057] Take 50 mg of muscle and grind it thoroughly using a hand grinder. Add 600 μL of tissue lysis buffer and 10 μL of proteinase K to a centrifuge tube, mix well, and place in a water bath at 55°C for 3 hours until the muscle tissue is completely dissolved. Gently shake once every 1 hour during the water bath. After the muscle is completely lysed, cool on ice for 5 minutes, add 200 μL of ammonium acetate solution (7.5 mol / L), mix thoroughly, and cool on ice for 5 minutes. After cooling, centrifuge at 12000 rpm for 10 minutes at 4°C, transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, gently mix by inverting, and let stand for 3 minutes. Centrifuge at 12000 rpm for 10 minutes at 4°C, discard the supernatant, and keep the precipitate. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm for 8 minutes, discard the supernatant, and keep the precipitate. Wash the precipitate again with 1 mL of 95% ethanol, centrifuge at 12000 rpm for 8 min, discard the supernatant, and retain the precipitate. Air dry thoroughly in a fume hood for 8 min, then dissolve in 200 μL of sterile ultrapure water. Assess DNA quality using 1.5% agarose gel electrophoresis and measure DNA concentration using a NanoDrop 2000 spectrophotometer. Dilute the sample DNA concentration to 5 ng / μL and store at -20℃ for subsequent genotyping.

[0058] (8) Genotyping of the three high-temperature resistant extreme phenotype populations obtained in step (1) was performed using KASP markers (population 1-Hsp70-1, population 2-Hsp70-1, population 3-Hsp70-1). The KASP system consisted of: 1.4 μL DNA, 0.075 μL each of primers F1 and F2, 0.2 μL primer R, 2.5 μL KASP-Mix, and 0.75 μL ddH2O. The KASP reaction program was: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 sec, 68℃ annealing for 1 min, 10 cycles, with the annealing temperature decreasing by 0.8℃ per cycle; 95℃ denaturation for 20 sec, 57℃ extension for 1 min, 34 cycles. After the reaction, the genotypes were analyzed using an LGC high-throughput genotyping instrument, and sequencing was performed simultaneously to verify the genotyping accuracy. The results showed that the KASP marker at one locus of the Hsp70-1 gene had good genotyping effect and high accuracy. The genotyping results are shown in the figure. Figure 4 As can be seen from A, the classification accuracy in this embodiment is approximately 90%.

[0059] Association analysis between genotype and heat resistance phenotype showed that the proportion of highly resistant individuals in the Hsp70-1-Hap1 haplotype combination was 81.48%, which was significantly higher than the proportion of susceptible individuals. The Hap1 haplotype was the dominant haplotype for heat resistance. In all three populations, the proportion of highly resistant individuals was higher than that of susceptible individuals. The heat resistance phenotype of Hsp70-1-Hap1 is shown in Table 6.

[0060] Table 6. Analysis of the proportion of Hsp70-1-Hap1 and high-temperature resistant phenotypes.

[0061]

[0062] Example 2

[0063] SNP site mining and heat resistance analysis of the Hsp70-2 gene:

[0064] (1) Obtaining the extreme heat-resistant phenotype population: In this experiment, three randomized groups were constructed, with a sample size of no less than 150 individuals in each group. The *Procambarus clarkii* was subjected to high-temperature (35℃) stress culture for 108 hours, and the survival time of each individual in the group was recorded. Based on the time of death, each individual in the above groups was classified into three phenotypes: Sensitive, Tolerant, and Strong Tolerant. Individuals that died between 0 and 36 hours were classified as Sensitive, those that died between 36 and 72 hours as Tolerant, and those that died between 72 and 108 hours as Strong Tolerant. The three phenotypes were repeatedly validated for each marked individual, resulting in three extreme heat-resistant phenotype populations: Population 1-Hsp70-2, Population 2-Hsp70-2, and Population 3-Hsp70-2.

[0065] (2) Extraction of hemolymph RNA from Procambarus clarkii, the specific steps are the same as in Example 1.

[0066] (3) cDNA was obtained by reverse transcription using a reverse transcription kit, and the specific steps were the same as in Example 1.

[0067] (4) Primers for the Hsp70-2 coding region were designed using Primer5 software based on data from the NCBI nucleic acid database. The primer sequences for the Hsp70-2 coding region are shown in Table 7.

[0068] Table 7. Primer sequence information for the Hsp70-2 coding region.

[0069]

[0070] (5) The Hsp70-2 coding region fragment was amplified using the above primers. The PCR system consisted of: 10 μL 2×PCR Mix, 1 μL each of F and R primers, 1 μL cDNA, and 7 μL ddH2O. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, for 35 cycles; 72℃ extension for 10 min. The size and quality of the PCR products were detected by 1.5% agarose gel electrophoresis. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared using the Sequencher software, which determined that there was a C>T synonymous mutation in the Hsp70-2 coding region of the thermoresistant gene at position NC_091241.1-14594131 in the chromosome.

[0071] (6) Based on the sequence information of the NCBI nucleic acid database, KASP molecular markers were developed for SNP sites. Primers were designed using Primer 5 software, and FAM and HEX fluorescent tags were added to the 5' ends of the F1 and F2 forward primers for the two genotypes, respectively. The underlined part of the sequence is a universal fluorescent tag. KASP marker information is shown in Table 3.

[0072] (7) Extract DNA from the muscle sample of Procambarus clarkii, using the same method as in Example 1.

[0073] (8) Genotyping of the three high-temperature resistant extreme phenotype populations obtained in step (1) was performed using KASP markers (population 1-Hsp70-2, population 2-Hsp70-2, population 3-Hsp70-2). The KASP system consisted of: 1.4 μL DNA, 0.075 μL each of primers F1 and F2, 0.2 μL primer R, 2.5 μL KASP-Mix, and 0.75 μL ddH2O. The KASP reaction program was: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 sec, 68℃ annealing for 1 min, 10 cycles, with the annealing temperature decreasing by 0.8℃ per cycle; 95℃ denaturation for 20 sec, 57℃ extension for 1 min, 34 cycles. After the reaction, the genotypes were analyzed using an LGC high-throughput genotyping instrument, and sequencing was performed simultaneously to verify the genotyping accuracy. The results showed that the KASP marker at one locus of the Hsp70-2 gene had good genotyping effect and high accuracy. The genotyping results are shown in the figure. Figure 4 As shown in B, the classification accuracy in this embodiment is approximately 90%.

[0074] Association analysis between genotype and heat resistance phenotype showed that the proportion of highly resistant individuals in the Hsp70-2-Hap1 haplotype combination was 81.29%, which was significantly higher than the proportion of susceptible individuals. The Hap1 haplotype was the dominant haplotype for heat resistance. In all three populations, the proportion of highly resistant individuals was significantly higher than that of susceptible individuals. The heat resistance phenotype of Hsp70-2-Hap1 is shown in Table 8.

[0075] Table 8. Phenotypic Analysis of Hsp70-2-Hap1 and High Temperature Resistance

[0076]

[0077] Example 3:

[0078] The combined analysis of heat-resistant haplotypes of the Hsp70-1 and Hsp70-2 genes was performed, and the specific steps were as follows:

[0079] (1) Obtaining a population with extreme high-temperature resistance phenotypes, the specific method is as follows:

[0080] A total of six high-temperature resistant extreme phenotype populations were constructed using Examples 1 and 2.

[0081] (2) Extraction of hemolymph RNA from Procambarus clarkii, the specific steps are the same as in Example 1.

[0082] (3) cDNA was obtained by reverse transcription using a reverse transcription kit, and the specific steps were the same as in Example 1.

[0083] (4) Primers were designed for the Hsp70-1 and Hsp70-2 coding regions using Primer5 software based on data from the NCBI nucleic acid database. Primer sequences are shown in Table 1.

[0084] (5) The Hsp70-1 and Hsp70-2 coding region fragments were amplified using the primers in Table 1. The PCR system consisted of 10 μL 2×PCRMix, 1 μL each of F and R primers, 1 μL cDNA, and 7 μL ddH2O. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, for 35 cycles; 72℃ extension for 10 min. The size and quality of the PCR products were detected by 1.5% agarose gel electrophoresis. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared using the Sequencher software, which determined that the Hsp70-1 coding region of the thermoresistant gene had a G>A synonymous mutation at the NC_091159.1-53803001 position in the chromosome. A C>T synonymous mutation was found in the coding region of the heat resistance gene Hsp70-2 at position NC_091241.1-14594131 of the chromosome.

[0085] (6) Based on the sequence information of the NCBI nucleic acid database, KASP molecular markers were developed for SNP sites. Primers were designed using Primer 5 software, and FAM and HEX fluorescent tags were added to the 5' ends of the F1 and F2 forward primers for the two genotypes, respectively. The underlined part of the sequence is a universal fluorescent tag. KASP marker information is shown in Table 3.

[0086] (7) Extract DNA from the muscle sample of Procambarus clarkii, using the same method as in Example 1.

[0087] (8) Genotyping of the nine high-temperature resistant extreme phenotype populations obtained in step (1) was performed using KASP markers. The KASP system consisted of: 1.4 μL DNA, 0.075 μL each of primers F1 and F2, 0.2 μL primer R, 2.5 μL KASP-Mix, and 0.75 μL ddH2O. The KASP reaction program was: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 sec, 68℃ annealing for 1 min, 10 cycles, with the annealing temperature decreasing by 0.8℃ per cycle; 95℃ denaturation for 20 sec, 57℃ extension for 1 min, 34 cycles. After the reaction, the genotypes were analyzed using an LGC high-throughput genotyping instrument, and sequencing was performed simultaneously to verify the genotyping accuracy. The genotyping results are shown in […]. Figure 4 As can be seen, the classification accuracy in this embodiment is approximately 90%.

[0088] (9) A combined analysis of two SNP loci of Hsp70-1 and Hsp70-2 revealed 59 individuals with extreme phenotypes in the dominant haplotype combination Hsp70-1-Hap1+Hsp70-2-Hap1. Although the number of individuals in the population is small, 91.53% of them are highly resistant, indicating great potential for application in the breeding of heat-resistant varieties. Specific combination information is shown in Table 9.

[0089] Table 9. Analysis of the High Temperature Resistance of the Dominant Haplotype Combination of Hsp70-1 + Hsp70-2

[0090]

[0091] (10) Two-gene combined analysis was performed on the two SNP loci of Hsp70-1 and Hsp70-2. A total of 85 individuals with extreme phenotypes in the Hsp70-1-Hap2+Hsp70-2-Hap2 inferior haplotype combination were found. Among them, 76 individuals had a susceptible phenotype, accounting for 89.41%; and 9 individuals had a strong resistant phenotype, accounting for only 10.59%. Specific combination information is shown in Table 10.

[0092] Table 10. Analysis of the High-Temperature Resistance of the Inferior Haplotype Combination of Hsp70-1 + Hsp70-2

[0093]

[0094]

[0095] References:

[0096] 1. Wei Qingshan. A Discussion on the Nomenclature of River (Crayfish) Species. Zoological Journal, 1989, 24: 50-51;

[0097] 2.Li RH,Bai SH,Yang DC,Dong CH.A crayfish Ras gene is involved in the defense ag ainstbacterial infection under high temperature.Fish and ShellfishImmunology,2018,86.

[0098] 3.Xi Zhu,Xin Ren,Lijing Xiong,Tiantian Liu,XufengBai,Geneticdissection of crayfish(Procambarus clarkii)high temperature tolerance andassessment of the potential application in breeding of the HSP genes,Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,Volume52,2024,101330.

Claims

1. A KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii*, characterized in that, The sequence of the primer combination is as follows:

2. A KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii*, characterized in that, The gene information corresponding to the primer combination is:

3. An application of a KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii*, characterized in that... Includes the following steps: (1) Obtaining the population with extreme high temperature resistance: The population was subjected to high temperature stress culture for 108 hours, and the mortality time of individuals in the population was counted. Based on the mortality time, the population was divided into three phenotypes: susceptible, resistant, and strongly resistant. The population with extreme high temperature resistance was obtained by screening. (2) Extraction of hemolymph RNA from all individuals of Procambarus clarkii obtained in step (1); (3) Obtain the cDNA corresponding to the RNA obtained in step (2) by reverse transcription and store it at -20℃ for later use; (4) Design and synthesize PCR primers. The primer sequence information is as follows: (5) Perform PCR amplification on the cDNA samples of the heat-resistant extreme phenotype individuals of Procambarus clarkii obtained in step (1); amplify the target fragment using the primers designed in step (4), and the amplified target fragment site information is as follows: (6) Develop KASP molecular markers for SNP sites, design KASP marker primers, and add FAM and HEX fluorescent tags to the 5' ends of the F1 and F2 forward primers for the two genotypes, respectively. The underlined part of the sequence is a universal fluorescent tag, so that the 3' end bases of the F1 and F2 forward primers correspond to the two alleles before and after the mutation, respectively; the KASP marker primer sequence information is as follows: (7) Extract DNA from all muscle samples of Procambarus clarkii, dilute the DNA concentration of each sample to 5 ng / μL, and store them at -20℃ for subsequent genotyping. (8) Use KASP markers to perform genotyping on the high-temperature resistant extreme phenotype population obtained in step (1): screen for haplotypes with superior high-temperature resistance; (9) Two SNP sites of Hsp70-1 and Hsp70-2 were analyzed by dual gene combination to screen for strong resistance genotype combinations.

4. The application of the KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii* according to claim 3, characterized in that, In step (1), individuals who died between 0 and 36 hours had a susceptible phenotype, those who died between 36 and 72 hours had an resistant phenotype, and those who died between 72 and 108 hours had a strongly resistant phenotype.

5. The application of the KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii* according to claim 3, characterized in that, Step (8) KASP reaction program is as follows: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 sec, 68℃ annealing for 1 min, 10 cycles, with the annealing temperature decreasing by 0.8℃ in each cycle; 95℃ denaturation for 20 sec, 57℃ extension for 1 min, 34 cycles.

6. The application of the KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii* according to claim 3, characterized in that, Step (8) The KASP system consists of: 1.4 μL DNA, 0.075 μL each of primers F1 and F2, 0.2 μL primer R, 2.5 μL KASP-Mix, and 0.75 μL ddH2O.

7. The application of the KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii* according to claim 3, characterized in that, Step (9) screened out the high-temperature resistant superior Cronobacter clarkii haplotypes Hsp70-1-Hap1 and Hsp70-2-Hap1.

8. The application of the KASP molecular marker primer combination for identifying the heat resistance of *Procambarus clarkii* according to claim 3, characterized in that, Step (9) screened and obtained the combined Hsp70-1 and Hsp70-2 genes of the red swamp crayfish with high temperature resistance.

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