Scylla paramamosain temperature regulation related functional gene microsatellite marker, detection method, kit, analysis method, screening method and breeding method

Abstract

The invention belongs to the technical field of biological information analysis, and particularly relates to a microsatellite marker, a detection method, a kit, an analysis method, a screening method and a breeding method of scylla paramamosain temperature regulation related functional genes. Through transcriptome data analysis, functional genes related to temperature adaptability are screened, the Scylla paramamosain temperature regulation related functional gene microsatellite marker is developed, polymorphic primers are provided, and a technical system and a detection method for Scylla paramamosain functional gene microsatellite marker development are established. A foundation is laid for genetic diversity research, genetic map construction and evolution analysis of the scylla paramamosain, and valuable and reliable molecular markers are provided for breeding of excellent varieties of the scylla paramamosain.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioinformatics analysis, and particularly relates to microsatellite markers, detection methods, kits, analysis methods, screening methods and breeding methods for temperature regulation-related functional genes of Scylla paramamosain. Background Art

[0002] Scylla paramamosain, as an important marine economic crab with significant body size advantages and fast growth characteristics in the family Portunidae, its growth and development process is significantly affected and regulated by environmental factors. Under the dual influence of global climate change and human activities, the habitat environment of Scylla paramamosain is undergoing significant changes, and the variation of temperature factors has significant ecological effects on its growth and development, reproductive strategies and survival adaptability.

[0003] Microsatellite, as short tandem repeat sequences commonly existing in eukaryotic genomes, has become an important molecular tool for population genetic diversity assessment and population structure analysis due to its advantages such as high polymorphism and genetic stability. In the international field of aquaculture breeding, the molecular marker-assisted selection technology based on microsatellite markers has developed into the core technology system of modern aquaculture genetics and breeding, showing extensive application value in the research of population genetic structure analysis and genetic improvement of aquatic animals.

[0004] Temperature, as a key environmental factor affecting the physiological metabolism and behavioral adaptation of poikilothermic animals, the research on its regulation mechanism is of great significance. When specific microsatellite markers are significantly associated with temperature tolerance traits, it may imply that these marker loci are closely related to the adaptive evolution of the population to environmental conditions. By identifying temperature-responsive related functional genes and developing their linked microsatellite markers, it can provide important genetic tools and theoretical basis for the molecular marker-assisted selection of stress-resistant varieties such as low-temperature tolerance and high-temperature tolerance of Scylla paramamosain.

[0005] The closest prior art is the phenotypic association marker method based on conventional Taq enzyme and single microsatellite locus amplification. This method uses universal SSR primers for simple typing of various marine crustaceans, and the typical representative is the technical route of amplifying one by one at a single locus and separating by gel electrophoresis. Although this technology can initially obtain polymorphic information, due to the fact that the amplification system and primer design do not fully consider the conservation of the surrounding sequences of the target gene and the variation characteristics of the repeat units, the amplification specificity is insufficient, and non-specific bands and primer dimer interference are likely to occur in the background of high-complexity genomes. In addition, this method does not conduct a collective study on temperature regulation functional genes and cannot directly establish a stable association with the temperature control phenotype. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides microsatellite markers for temperature regulation-related functional genes of Scylla paramamosain and a detection method thereof. Based on bioinformatics analysis, temperature regulation-related gene information is obtained through database retrieval, and compared with the transcriptome data of Scylla paramamosain to screen out candidate genes related to temperature response. The SSRhunter software is used to scan the microsatellite loci of the target gene sequence, and finally 28 temperature regulation-related functional gene sequence fragments containing microsatellite loci are identified. The Primer Premier 5 software is used to design specific primers for the above 28 microsatellite loci. After verification, a total of 27 pairs of effective primers are obtained. The genomic DNA of Scylla paramamosain is amplified by PCR, and 17 pairs of primers can stably amplify the expected bands. The amplification products are further sequenced, and 7 microsatellite markers with significant polymorphism are screened out. To improve the detection throughput, the polymorphic primers are fluorescently labeled after single-strand sequence detection and PCR amplification is carried out in a larger-scale population sample. After the amplification products are separated by capillary electrophoresis, the GeneMapper software is used for genotyping, and finally 7 highly polymorphic microsatellite markers for temperature regulation-related functional genes of Scylla paramamosain are obtained.

[0007] The present invention is realized as follows. A microsatellite marker for temperature regulation-related functional genes of Scylla paramamosain, the 7 microsatellite markers are respectively named: NC-04 (ATP), NC-15 (CRU), NC-17 (FOU), NC-13 (MYO), NC-21 (NAS), NC-05 (RAC) and NC-25 (TRO).

[0008] Furthermore, the specific primers of the microsatellite markers include:

[0009] Specific primers for microsatellite locus NC-04 (ATP): F: SEQ ID NO1: 5’-CGGAGTCCTCAATACGG-3', R: SEQ ID NO2: 5’-CCTAACATCTGCAACGAAT-3’;

[0010] Specific primers for microsatellite locus NC-15 (CRU): F: SEQ ID NO3: 5’-GGAGTTGTGGCACTGAAA-3', R: SEQ ID NO4: 5’-ATGGCGAGGATGTCAATA-3’;

[0011] Specific primers for microsatellite locus NC-17 (FOU): F: SEQ ID NO5: 5’-TGTCTGGTTTGTGGGTGTTA-3', R: SEQ ID NO6: 5’-AAGAGATAGGCAGACGATTAGA-3’;

[0012] Specific primers for microsatellite locus NC-13 (MYO): F: SEQ ID NO7: 5’-TAGCCAGCCTGTCCTTCA-3', R: SEQ ID NO8: 5’-GTGAGGGTGGTGATAGGGA-3’;

[0013] Specific primers for microsatellite locus NC-21 (NAS): F: SEQ ID NO9: 5’-GGAAGTGTTGTGCTGGTG-3', R: SEQ ID NO10: 5’-ATGGCGAGGATGTCAATA-3’;

[0014] Specific primers for microsatellite locus NC-05 (RAC): F: SEQ ID NO11: 5’-GCATACACTGGCATTGGG-3', R: SEQ ID NO12: 5’-CTAATGACTCCAGGACTTTGC-3’;

[0015] Specific primers for microsatellite locus NC-25 (TRO): F: SEQ ID NO13: 5’-GAAACGGCGTAAGGACAC-3', R: SEQ ID NO14: 5’-GAGCGAACAAGAGCAGGA-3’.

[0016] Another object of the present invention is to provide a detection method for microsatellite markers of temperature regulation-related functional genes of Scylla paramamosain, which specifically includes the following steps:

[0017] S1, Extract the genomic DNA of Scylla paramamosain;

[0018] S2, Using the genomic DNA extracted in S1 as the PCR amplification template, and respectively performing PCR amplification using the specific primers of the microsatellite markers of the temperature regulation-related functional genes of Scylla paramamosain described above;

[0019] S3, Use capillary electrophoresis technology to genotype the PCR amplification products.

[0020] Furthermore, for the PCR amplification in S2, the reaction system is: 2×TaqPCR Mix 12.5 μL, 100 nM forward primer 1 μL, 100 nM reverse primer 1 μL, DNA template 2 μL, ddH2O 8.5 μL, totaling 25 μL.

[0021] Furthermore, for the PCR amplification in S2, the amplification reaction procedure is as follows: pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 15 s, annealing at the annealing temperature of the microsatellite marker specific primer set related to the temperature regulation functional gene of Scylla paramamosain for 15 s, extension at 72 °C for 30 s, with a total of 30 cycles, and finally extension at 72 °C for 10 min; the annealing temperatures of the microsatellite marker specific primer set related to the temperature regulation functional gene of Scylla paramamosain are respectively: microsatellite locus NC-04 (ATP): 50 °C, microsatellite loci NC-15 (CRU) and NC-21 (NAS): 51 °C, microsatellite loci NC-17 (FOU), NC-05 (RAC) and NC-25 (TRO): 54 °C, microsatellite locus NC-13 (MYO): 55 °C.

[0022] Combined with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0023] Using the specific primers of the microsatellite markers related to the temperature regulation functional gene of Scylla paramamosain of the present invention to detect 30 Scylla paramamosain samples respectively, the results show that: all 7 microsatellite markers have high polymorphism, indicating that the 7 microsatellite markers related to the temperature regulation functional gene of Scylla paramamosain of the present invention can be effectively used for the analysis of the population genetic structure of Scylla paramamosain and molecular marker-assisted breeding, especially having important application value in the breeding of temperature-resistant excellent varieties of Scylla paramamosain.

[0024] All 7 microsatellite markers developed by the present invention are located in the sequence regions of the temperature regulation functional genes of Scylla paramamosain, and these genes play important roles in the growth, development and environmental adaptation processes of crustaceans. Therefore, these 7 microsatellite markers have important application value in the breeding of temperature-tolerant excellent varieties of Scylla paramamosain. By analyzing the transcriptome data, the present invention screens out the functional genes related to temperature adaptability, develops the microsatellite markers related to the temperature regulation functional gene of Scylla paramamosain, provides polymorphic primers, and establishes a technical system and detection method for the development of microsatellite markers of functional genes of Scylla paramamosain, which will not only lay a foundation for the research on the genetic diversity, construction of genetic maps and evolutionary analysis of Scylla paramamosain, but also provide valuable and reliable molecular markers for the breeding of excellent varieties of Scylla paramamosain. Description of the Drawings

[0025] Figure 1 is the flow chart of the detection method of the microsatellite markers related to the temperature regulation functional gene of Scylla paramamosain provided by the embodiment of the present invention;

[0026] Figure 2 is the fluorescence-labeled peak map of the microsatellite locus provided by the embodiment of the present invention. Detailed Embodiments

[0027] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] In view of the high variability and polymorphism characteristics of the population temperature-controlled phenotypes of Scylla paramamosain, the present invention first screened out seven microsatellite loci (NC-04, NC-15, NC-17, NC-13, NC-21, NC-05, NC-25) closely related to temperature regulation functions through whole-genome resequencing and tandem repeat sequence mining, effectively filling the technical gap of the scarcity of molecular markers for existing temperature-controlled traits.

[0029] After the loci were confirmed, a conservative region localization strategy based on multiple sequence alignment was adopted to design highly specific oligonucleotide primers, ensuring that each primer pair annealed and bound only to the target microsatellite repeat sequence and its upstream and downstream specific regions, avoiding non-specific amplification and primer dimer formation, thereby improving the amplification accuracy and repeatability.

[0030] The amplified products were analyzed by fluorescence-labeled capillary electrophoresis. The high-resolution capillary system was used to accurately genotype microsatellite fragments of different lengths. The detection data was analyzed by an automated software to resolve the peak patterns and perform genotype normalization, ensuring comparable allele length information under the same electrophoresis conditions.

[0031] Combined with population genetics statistical software, quantitative analysis was performed on the obtained allele frequencies, heterozygosity, and polymorphic information content, and linkage disequilibrium and association analysis methods were used to screen out marker loci significantly related to high-temperature or low-temperature tolerance traits, realizing accurate association from the molecular level to the phenotypic level.

[0032] Integrating the above markers and detection processes into molecular breeding, through a strategy combining background selection and trait selection, high-temperature tolerance excellent families were quickly identified in the breeding population, accelerating the breeding of new strains and reducing the breeding cycle, providing an efficient and reliable molecular-assisted decision-making tool for the industrialized farming of Scylla paramamosain.

[0033] As Figure 1 shown, the method for detecting microsatellite markers of genes related to temperature regulation in Scylla paramamosain specifically includes the following steps:

[0034] S1, extracting genomic DNA of Scylla paramamosain;

[0035] S2, using the genomic DNA extracted in S1 as the PCR amplification template, and respectively performing PCR amplification using the specific primers of the microsatellite markers of the genes related to temperature regulation in Scylla paramamosain as described above;

[0036] S3, typing the PCR amplification products by capillary electrophoresis technology.

[0037] Furthermore, for the PCR amplification in S2, the reaction system is as follows: 12.5 μL of 2×TaqPCR Mix, 1 μL of 100 nM forward primer, 1 μL of 100 nM reverse primer, 2 μL of DNA template, and 8.5 μL of ddH2O, with a total of 25 μL.

[0038] Furthermore, for the PCR amplification in S2, the amplification reaction procedure is as follows: pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 15 s, annealing at the annealing temperature of the microsatellite marker specific primer set related to the temperature regulation functional genes of Scylla paramamosain for 15 s, extension at 72 °C for 30 s, with a total of 30 cycles, and finally extension at 72 °C for 10 min; the annealing temperatures of the microsatellite marker specific primer set related to the temperature regulation functional genes of Scylla paramamosain are respectively: microsatellite locus NC-04 (ATP): 50 °C, microsatellite loci NC-15 (CRU) and NC-21 (NAS): 51 °C, microsatellite loci NC-17 (FOU), NC-05 (RAC) and NC-25 (TRO): 54 °C, microsatellite locus NC-13 (MYO): 55 °C.

[0039] 1. Screening of temperature-related functional genes of Scylla paramamosain and search for microsatellite loci

[0040] Based on bioinformatics analysis, temperature regulation-related gene information is obtained through database retrieval, and then the sequences of temperature regulation-related functional genes are obtained from the Scylla paramamosain transcriptome library. The SSRhunter software is used to scan the microsatellite loci of the screened temperature regulation-related functional gene sequences, and the screening criteria are set as follows: dinucleotide repeats n≥5 times, trinucleotide repeats n≥4 times, and tetranucleotide repeats n≥3 times. Through further bioinformatics analysis, 28 temperature regulation-related functional gene sequences containing microsatellite loci are finally identified.

[0041] 2. Design of microsatellite markers primers for temperature regulation-related functional genes of Scylla paramamosain

[0042] Based on the SSR loci in the 28 temperature regulation-related functional gene sequences of Scylla paramamosain obtained by screening, the Primer Premier5 software is used in the present invention to design the primers for microsatellite markers. The parameter settings for primer design are as follows: primer length 18 - 24 bp, GC content 40 - 60%, Tm value 50 - 60 °C, and the difference in Tm values between the upstream and downstream primers is not more than 5 °C. At the same time, software analysis is used to avoid adverse factors such as primer dimers, hairpin structures, and mismatches, ensuring that the length of the amplification product is controlled within the range of 100 - 500 bp. After strict screening, 27 pairs of specific primers are finally successfully designed and synthesized.

[0043] 3. Screening and Result Analysis of Microsatellite Marker Primers for Function Genes Related to Temperature Regulation in Scylla paramamosain

[0044] 3.1 Extraction of Genomic DNA from Scylla paramamosain

[0045] In this invention, wild Scylla paramamosain populations from Zhejiang, Guangdong, and Hainan were selected as experimental objects. 10 individuals were randomly collected from each geographical population, with a total of 30 samples. After all samples were collected, they were immediately stored on ice and transported back to the laboratory within 24 hours. 30 mg of the swimming leg muscle tissue of each sample was taken, and the genomic DNA of Scylla paramamosain was extracted using a genomic DNA extraction kit for marine animal tissues (Tiangen Biochemical Technology Co., Ltd., Beijing). The specific operation steps were carried out strictly according to the instructions. After the genomic DNA extraction was completed, a preliminary quality assessment was performed by 1.0% agarose gel electrophoresis (voltage 100 V, 20 min), using Hind III digest DNA Marker as the molecular weight standard. Qualified genomic DNA samples showed clear main bands without obvious degradation.

[0046] 3.2 Preliminary Screening of Microsatellite Primers

[0047] 10 samples were randomly selected from the above-extracted genomic DNA of Scylla paramamosain as PCR amplification templates, and 27 pairs of primers were used to perform gradient PCR amplification on this genomic DNA. The reaction system was: 2×TaqPCR Mix 12.5 μL, 100 nM forward primer 1 μL, 100 nM reverse primer 1 μL, DNA template 2 μL, ddH2O 8.5 μL, totaling 25 μL. The reaction program was: pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 15 s, annealing at the annealing temperature of the microsatellite marker specific primer set related to the temperature regulation of Scylla paramamosain for 15 s (as shown in Table 1), extension at 72 °C for 30 s, 30 cycles, and finally extension at 72 °C for 10 min. The PCR amplification products were detected by 2% agarose electrophoresis (100 V, 20 min), showing that 17 pairs of primers could stably amplify clear bands at specific annealing temperatures. After sequencing and analysis of the amplification products, the results showed that all 17 pairs of primers could amplify the target bands.

[0048] Table 1 Information of 7 Highly Polymorphic Microsatellite Markers

[0049]

[0050] 3.3 Screening of Polymorphic Primers

[0051] The amplification products of 17 groups of specific primer pairs were sequenced and typed, and preliminary polymorphism analysis was carried out on the target sequences. Based on the number of repeats of the microsatellite sequences, 7 microsatellite markers of Scylla paramamosain temperature regulation-related functional genes with polymorphism were screened out.

[0052] 3.4 Fluorescent Labeling Typing and Result Analysis

[0053] The 5' ends of the upstream primers of the 7 pairs of primers with polymorphism screened out were fluorescently labeled with FAM fluorescent dye, and PCR amplification was carried out using the genomic DNA of 30 Scylla paramamosain extracted above as the template. The reaction system was the same as that in the preliminary screening of microsatellite primers in step 3.2 above, and the reaction program was the same as that in step 3.2 above except for the annealing temperature. The annealing temperature corresponding to each primer was referred to Table 1. The PCR products were sent to Shanghai Jieli Biotechnology Co., Ltd. for typing, and the GeneMapper software was used to interpret the specific values of the allele fragments, and 7 microsatellite markers of Scylla paramamosain temperature regulation-related genes with polymorphism were determined. Their loci were named: NC-04 (ATP), NC-15 (CRU), NC-17(FOU), NC-13 (MYO), NC-21 (NAS), NC-05 (RAC) and NC-25 (TRO).

[0054] The expected heterozygosity and observed heterozygosity were calculated using the fstat software, and the polymorphic information content (PIC) was calculated using the PIC Calc software. The results showed that the number of alleles of the above 7 polymorphic Scylla paramamosain temperature regulation-related functional gene microsatellite markers were 6, 9, 5, 6, 7, 7 and 9 respectively; the observed heterozygosities were 0.583, 0.636, 0.417, 0.818, 0.583, 0.667 and 0.364 respectively; the expected heterozygosities were 0.807, 0.900, 0.769, 0.823, 0.761, 0.841 and 0.909 respectively; the PIC values were 0.7283, 0.8295, 0.6844, 0.7536, 0.6909, 0.7732 and 0.8254 respectively (as shown in Table 2), all of which were greater than 0.5, indicating that these 7 microsatellite markers all had high polymorphism and could be applied to the analysis of the population genetic structure of Scylla paramamosain and molecular marker-assisted breeding.

[0055] Table 2 Genetic Information Parameters of 7 Highly Polymorphic Microsatellite Markers

[0056]

[0057] NC-04 (ATP):

[0058] SEQ ID NO15:

[0059] CGGAGTCCTCAATACGGAGCGGAGGGCGGCGGCACCAGTCAGTCCTGAGCACCATGGCCAGCCAGTCGCAGGGAGTCCAGCAGCTGCTCACCGCCGAGAAAAAGGCAGCTGAGAAGGTCGCAGAGGCCAGGAAACGTAAGAATTAAGCACTATATACATTATCCTTCCTATACTGGTAGTTGTAATGAATTGTCACGCGTGTAGGAGAAGATATGAGAGAGGGTTTAAAGGGACAGGTTTAAAGAGCTTATTTTGTGTTTTGGTTATGTTTATTGGTTATTTTCTTTCGTTTTGTATAGTGGAGTGTTTGTGGGTGTGTGTTTTTGTAGGTCATGTTTGTTTCTGTGTGTGTGTTTTTTTTTTGTGTGTGTGCCATTTTTATTGGTAATGGTGATTGTTGTGTGTTATTGATTCGTTGCAGATGTTAGG

[0060] NC-15 (CRU):

[0061] SEQ ID NO16:

[0062] GGAGTTGTGGCACTGAAACAATAAGGTCATATATTGCAGGGTGAGATTACGAGAAAAGGGTGTTACTTCATGTAGTGGTAATGCACTACTCACCGCAGAACCGTATTAGTGGTGATGTTGCTGCTTGAGTTGATAATAAACATATATAGAAGGGTGAGTCGGAGGGGGAGGAAAAGGAGCACACACACACACACACACACACACACACACACACGCAAATTATACCTGAGCGTGTCTGATTACACCATCAGCCGCATGGACTCGTCTGCGGCTACGCGACACACTTGTTATTAGTCCAGATTAGCGGCGACTGGCGACCTAATACTGTAGGTGTATTGTCAATTATTGACATCCTCGCCAT

[0063] NC-17 (FOU):

[0064] SEQ ID NO17:

[0065] TGTCTGGTTTGTGGGTGTTACTTTGATTGTTTTCTTGTTACTTTTGATGTTTATGCACTACTACTACTGTGTGTGTGTGTGTGTGAGGTAAGCATTTAGTCAGTTACTGCAGATAAGTTAATCAGTAAGGCAATTAGTCAAAGTAGAAAGATGCATTATCTGTCTGTTTATTATCCTGTGTATGTATGTATGTATCTGTTCATTTATCTGTCATTTGGGTATCTGTCTATCTGTCTATCTATTTGTGTATATTTGTCTATCCATCTTTCTGTCTATCCGTCTTTGTCTATCCGTCCGTATGTTTGTATTAGTGTCTGTCCGTCGGTCTATGAACGTCTAATCGTCTGCCTATCTCTT

[0066] NC-13 (MYO):

[0067] SEQ ID NO18:

[0068] TAGCCAGCCTGTCCTTCAAGTCGTGGCCAAATAGCAGCCAATCGTAGGCCACAGTCAGGTAGAGAATCTGTGAGGAGCGTAGAGTAGTTAGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTAATCTCAATTGCAGTAACGACAAAGGTACGACAATCTCTCTCTCTCTCTCTCTCTCTCTCTATCATCCCTATCACCACCCTCAC

[0069] NC-21 (NAS):

[0070] SEQ ID NO19:

[0071] GGAAGTGTTGTGCTGGTGGCTGACGGTGTGGAATATGATGGCGTGCTGGGGAGGGGTTGGAGGGGACGGGAAGGAGCAGGCGTGGGCTAGACTAGACTGGATGAGATTTTTTTTTTATTTTTTATTTTTTTTTTTAAGGACAGACAATTAACGAGTATTTTTTTTCATTTTATTGCTGAGTTATTTATTTATTTATTTATTTATTTTTGTGCCCCATTGGAAAGGACGAGAGGTGGTAAAGTTAGTAATAATCTCTTTTCTTCGAATGGATGTGTACGTGACGGTAAATGATGTGCACCTACAAGTATGAGATGCTGCGATTT

[0072] NC-05 (RAC):

[0073] SEQ ID NO20:

[0074] GCATACACTGGCATTGGGTCCTGATGTACACTAACTAAAAAAAAAGTCCACCTACACTTTCTCAACTTCCTGTGCCCTTGATGCTGTCAGCCCTCTCACCCCACAATTCACACATTTTATGAGAGGACGGGATGATCAAGAGGAGATGAGGTAGGTAAGTCAGAGGTAGATAAAGTAGTTATTCCTTAAGGATTGTCAGCAGCTATGAAGAATTCCACTTTCAATAAAGAGCCTCCCAGCAACAAGGTGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCAGTCTCTCTCCACCTGGATTTGTTTCCCCACAGGAACTCTACTGCCAAAAGGCATATAGGGCAAAGTCCTGGAGTCATTAG

[0075] NC-25 (TRO):

[0076] SEQ ID NO21:

[0077] GAAACGGCGTAAGGACACCTGTATTATTAAAACGACGTTAAGAATCTCTCTCTCTCTCTCTCTCTCCTCTCTCTCTCTCTCTCTCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCACTGATAGTGCTAGTCCCTGCAGAGTATAGTTTGTTAGAATGTTTGGTAGAAGTCAGCCATTTTGTCACTGAGCTCGATTGAGTTGAGGCATAGTGAAACAGGAAAATGAATGTTCTCAAAGTTAGTTTGTTAGTGGTTTAGGAGTGAATCTTTAAGTAGTCTGTGCATGAATTATTATGAAAGAGGGGTAAGGCCTGTGTGGTGATCCTGCTCTTGTTCGCTC

[0078] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. Microsatellite markers for Scylla paramamosain temperature regulation-related functional genes, including seven microsatellite markers, namely NC-04 ATP, NC-15 CRU, NC-17 FOU, NC-13 MYO, NC-21 NAS, NC-05 RAC, and NC-25 TRO.

2. The microsatellite marker according to claim 1, wherein Specific primers are designed for the seven microsatellite markers respectively, and the primer sequences are as follows: Forward primer for NC-04 ATP locus: 5′-CGGAGTCCTCAATACGG-3′, Reverse primer: 5′-CCTAACATCTGCAACGAAT-3′ Forward primer for NC-15 CRU locus: 5′-GGAGTTGTGGCACTGAAA-3′, Reverse primer: 5′-ATGGCGAGGATGTCAATA-3′ Forward primer for NC-17 FOU locus: 5′-TGTCTGGTTTGTGGGTGTTA-3′, Reverse primer: 5′-AAGAGATAGGCAGACGATTAGA-3′ Forward primer for NC-13 MYO locus: 5′-TAGCCAGCCTGTCCTTCA-3′, Reverse primer: 5′-GTGAGGGTGGTGATAGGGA-3′ Forward primer for NC-21 NAS locus: 5′-GGAAGTGTTGTGCTGGTG-3′, Reverse primer: 5′-ATGGCGAGGATGTCAATA-3′ Forward primer for NC-05 RAC locus: 5′-GCATACACTGGCATTGGG-3′, Reverse primer: 5′-CTAATGACTCCAGGACTTTGC-3′ Forward primer for NC-25 TRO locus: 5′-GAAACGGCGTAAGGACAC-3′, Reverse primer: 5′-GAGCGAACAAGAGCAGGA-3′.

3. A detection method for microsatellite markers of functional genes related to temperature regulation in Scylla paramamosain, characterized in that, It includes the following steps: S1 Extract the genomic DNA of Scylla paramamosain; S2 Using the genomic DNA obtained in S1 as the PCR amplification template, and amplify it using the specific primers described in claim 2; S3 Perform capillary electrophoresis typing on the PCR amplification product.

4. The detection method according to claim 3, wherein The PCR amplification reaction system consists of the following components by volume ratio: 2×Taq PCR Mix 12.5 μL, forward primer 100 nM 1 μL, reverse primer 100 nM 1 μL, DNA template 2 μL, purified water to 25 μL.

5. The detection method according to claim 3, wherein The PCR amplification program was as follows: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 15 s; annealing for 15 s; extension at 72 °C for 30 s; 30 cycles; final extension at 72 °C for 10 min; the annealing temperatures were: 50 °C for the NC-04 ATP site; 51 °C for the NC-15 CRU site; 54 °C for the NC-17 FOU site; 55 °C for the NC-13 MYO site; 51 °C for the NC-21 NAS site; 54 °C for the NC-05 RAC site; 54 °C for the NC-25 TRO site.

6. A genotyping kit based on the microsatellite marker described in claim 1, characterized in that, The kit includes specific primers, Taq PCR Mix, capillary electrophoresis detection buffer, and primer storage buffer.

7. The kit according to claim 6, wherein The Taq PCR Mix is in a pre-packaged format and contains DNA polymerase, dNTP, and reaction buffer.

8. A method for analyzing the population genetic diversity of Scylla paramamosain based on the microsatellite markers described in claim 1, characterized in that, After genotyping is completed according to the detection method described in claim 3, allele frequencies, gene diversity, and polymorphism information indices are calculated using genetic statistical software.

9. A screening method for the heat tolerance breeding materials of Scylla paramamosain based on the microsatellite markers described in claim 1, characterized in that, Samples of Scylla paramamosain in different temperature treatment groups are genotyped, and correlation analysis is performed based on the genotyping results and temperature tolerance phenotypic data to determine molecular markers significantly associated with temperature tolerance traits.

10. A method for molecular marker-assisted breeding of Scylla paramamosain, characterized in that, It includes the following steps: The method described in claim 8 is used for background selection in the breeding population; The method described in claim 9 is used for selection of temperature tolerance traits in target breeding.

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