Application of SSR (simple sequence repeat) labeled detection kit in genetic feature analysis of sparus nipponensis

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to application of an SSR (simple sequence repeat) labeled detection kit in genetic feature analysis of sparus nipponensis. The SSR marker is selected from one or more of AS5-4, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12 and AS6-3, and the SSR marker is selected from one or more of AS5-4, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS3-7, AS6-4, AS5-10, AS5-2, AS5-12 and AS6-3. The SSR marker is used for constructing a nine-fold fluorescent PCR combination and a ten-fold fluorescent PCR combination, so that multi-site genotype identification can be realized, the detection efficiency can be obviously improved, the detection cost can be reduced, false positive phenomena and personal errors can be reduced, the time can be saved to the greatest extent, and the method is suitable for large-scale popularization and application. The method is the first choice for enhancement and release genetic effect evaluation, individual / family identification, family traceability analysis and germplasm resource evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the application of an SSR marker detection kit in the genetic characteristic analysis of black sea bream, particularly for genetic characteristic analysis of wild black sea bream populations in the early stage of stock enhancement and release. Background Technology

[0002] Black sea bream Acanthopagrus schlegelii Black sea bream, also known as red snapper, is a nearshore warm-water bottom-dwelling fish with advantages such as rapid growth, wide diet, salt tolerance, wide temperature tolerance, delicious flesh, small range of movement, and strong disease resistance. In recent years, due to overfishing, habitat destruction, and deterioration of the fishery ecological environment, wild black sea bream resources have already suffered severe decline, affecting the sustainability of fishery resources. To restore black sea bream resources and promote the sustainable use of fishery resources, a combination of natural propagation of wild resources and artificial breeding and release of seedlings has become the main restoration measure. However, current black sea bream propagation and release is based on an extensive approach of expanding scale, with propagation goals focusing more on increasing resource quantity and lacking research on the impact of propagation activities on the genetic characteristics of natural germplasm, i.e., ignoring the assessment of genetic risks of propagation and release. This is incompatible with the original intention of fishery resource conservation aimed at population restoration and the requirements of sustainable fishery development goals.

[0003] Numerous studies have shown that due to the limited effective parent population, multiple generations of breeding, origin from different waters, or belonging to different populations, there are usually significant genetic differences between artificially bred seedlings and wild populations. Large-scale stocking and release will disrupt the gene pool of wild populations through hybridization, affecting the expression of local adaptive genomes in wild individuals, altering the genetic composition of the population, reducing the level of genetic variation, and triggering genetic homogenization. This, in turn, reduces the level of genetic differentiation, genetic fitness, and health, leading to a series of genetic risks.

[0004] To reduce genetic risks and maximize the benefits of stock enhancement, some scholars believe that genetic risks should be effectively controlled and assessed from the following two aspects: First, the genetic risks caused by stock enhancement should be assessed at the source. This means conducting a genetic baseline survey of the wild populations of the stocked species, the stocked seedlings, and their breeding parents before stock enhancement, so as to ensure that the genetic background of the stocked population matches the wild population as closely as possible, thereby reducing the negative genetic impact of stock enhancement on the wild population's gene pool. Second, during the stock enhancement process, dynamic monitoring of the genetic risks of stock enhancement should be conducted to assess the impact of the released population on the genetic characteristics of the wild population, and this monitoring should be implemented throughout the entire stock enhancement activity to scientifically guide stock enhancement from a genetic perspective. In summary, effectively controlling and assessing the genetic risks of stock enhancement before and after stock enhancement is essential for the scientific and orderly development of stock enhancement and the effective management of genetic resources. Therefore, conducting genetic characteristic analysis and assessing the genetic risks of stock enhancement for black sea bream before and after stock enhancement is crucial. Summary of the Invention

[0005] In view of this, the present invention utilizes SSR markers with high genetic recognition in black sea bream to achieve efficient genotyping of large-scale samples, thereby assessing the genetic risk of black sea bream stock enhancement and release.

[0006] The specific technical solution provided by this invention is as follows: This invention provides an application of an SSR marker detection kit in the genetic characteristic analysis of black sea bream, wherein the SSR marker is selected from any one or a combination of several of AS5-4, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, and AS6-3; The repeating unit of AS5-4 is TCTAT, and the number of repetitions is 6. The repeating unit of AS6-6 is CGATGG, and the number of repetitions is 5. The repeating unit in AS4-6 is TCTA, and the number of repetitions is 15. AS5-9 has a repeating unit of GTGTT and a repeat count of 6. The repeating unit in AS6-8 is AGGAGA, and the number of repetitions is 5. AS3-6 uses the TTG as its repeating unit and repeats 8 times. The repeating unit of AS4-4 is GAA, and the number of repetitions is 5. The repeating unit of AS3-15 is AGA, and the number of repetitions is 12. The repeating unit of AS3-7 is TCA, and the number of repetitions is 8. The repeating unit of AS3-3 is CTT, and the number of repetitions is 18. The repeating unit in AS5-8 is TAGAA, and the number of repetitions is 9. The repeating unit of AS6-5 is AGGGTT, and the number of repetitions is 5. The repeating unit of AS3-14 is AAT, and the number of repetitions is 8. The repeating unit of AS4-7 is TTCT, and the number of repetitions is 9. The repeating unit in AS6-4 is CCTGGT, and the number of repetitions is 5. The repeating unit of AS5-10 is TTGTT, and the number of repetitions is 5. AS5-2's repeating unit is AACAT, and the number of repetitions is 7; The repeating unit of AS5-12 is TTAGA, and the number of repetitions is 9; The repeating unit in AS6-3 is CAAGTA, and the number of repetitions is 5.

[0007] In a second aspect, the present invention provides a primer pair for detecting the SSR marker, the sequence of which is as follows: The forward primer sequence for detecting AS3-6 is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The forward primer sequence for detecting AS3-7 is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The forward primer sequence for detecting AS3-15 is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; The forward primer sequence for detecting AS4-4 is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8; The forward primer sequence for detecting AS4-6 is shown in SEQ ID NO.9, and the reverse primer sequence is shown in SEQ ID NO.10; The forward primer sequence for detecting AS5-4 is shown in SEQ ID NO.11, and the reverse primer sequence is shown in SEQ ID NO.12; The forward primer sequence for detecting AS5-9 is shown in SEQ ID NO.13, and the reverse primer sequence is shown in SEQ ID NO.14; The forward primer sequence for detecting AS6-6 is shown in SEQ ID NO.15, and the reverse primer sequence is shown in SEQ ID NO.16. The forward primer sequence for detecting AS6-8 is shown in SEQ ID NO.17, and the reverse primer sequence is shown in SEQ ID NO.18; The forward primer sequence for detecting AS3-3 is shown in SEQ ID NO.19, and the reverse primer sequence is shown in SEQ ID NO.20; The forward primer sequence for detecting AS3-14 is shown in SEQ ID NO.21, and the reverse primer sequence is shown in SEQ ID NO.22; The forward primer sequence for detecting AS4-7 is shown in SEQ ID NO.23, and the reverse primer sequence is shown in SEQ ID NO.24; The forward primer sequence for detecting AS5-2 is shown in SEQ ID NO.25, and the reverse primer sequence is shown in SEQ ID NO.26; The forward primer sequence for detecting AS5-8 is shown in SEQ ID NO.27, and the reverse primer sequence is shown in SEQ ID NO.28; The forward primer sequence for detecting AS5-10 is shown in SEQ ID NO.29, and the reverse primer sequence is shown in SEQ ID NO.30; The forward primer sequence for detecting AS5-12 is shown in SEQ ID NO.31, and the reverse primer sequence is shown in SEQ ID NO.32; The forward primer sequence for detecting AS6-3 is shown in SEQ ID NO.33, and the reverse primer sequence is shown in SEQ ID NO.34; The forward primer sequence for detecting AS6-4 is shown in SEQ ID NO.35, and the reverse primer sequence is shown in SEQ ID NO.36; The forward primer sequence for detecting AS6-5 is shown in SEQ ID NO.37, and the reverse primer sequence is shown in SEQ ID NO.38.

[0008] In a preferred embodiment of the present invention, the 5' end of the forward primers for amplifying AS3-6, AS4-4, AS5-2, and AS5-10 is marked with a fluorescent label ROX; The forward primers for amplifying AS3-7, AS3-15, AS5-12, and AS6-3 have a fluorescent label TAMRA at their 5' ends; The forward primers for amplifying AS4-6, AS5-9, AS6-8, AS3-14, AS4-7, and AS6-4 have a 5' end labeled with HEX fluorescently. The forward primers for amplifying AS5-4, AS6-6, AS3-3, AS5-8, and AS6-5 have a fluorescent label FAM at their 5' ends.

[0009] In a third aspect, the present invention provides a kit for genetic characteristic analysis of black sea bream, the kit comprising the primer pair described above.

[0010] In a fourth aspect, the present invention provides the use of the SSR marker, the primer pair, or the kit in the genetic characterization of black sea bream.

[0011] As a preferred embodiment of the present invention, the SSR marker, the primer pair, or the kit is used to conduct a genetic baseline survey of wild black sea bream populations before the release of aquatic organisms.

[0012] In a fifth aspect, the present invention provides a method for analyzing the genetic characteristics of black sea bream, characterized by comprising the following steps: Extract whole-genome DNA from the sample to be tested; The primer pair was used to amplify the whole genome DNA by PCR; The amplified products were subjected to capillary electrophoresis, and the genetic characteristics of the black sea bream were analyzed based on the electrophoresis results. The primer pairs are composed of amplification primers of AS3-6, AS3-7, AS3-15, AS5-4, AS4-4, AS4-6, AS5-9, AS6-6 and AS6-8, or are composed of amplification primers of AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12 and AS6-3.

[0013] In a preferred embodiment of the present invention, the mixing volume ratio of the amplification primers of AS3-6, AS3-7, AS3-15, AS4-4, AS4-6, AS5-4, AS5-9, AS6-6 and AS6-8 is 5∶9∶9∶7∶3∶3∶2∶3∶2, and the forward and reverse primers are mixed in equal proportions. The mixing volume ratio of the amplification primers for AS3-3, AS3-14, AS4-7, AS5-2, AS5-8, AS5-10, AS5-12, AS6-3, AS6-4, and AS6-5 is 1:3:2:6:2:2:8:9:2, and the forward and reverse primers are mixed in equal proportions.

[0014] In a preferred embodiment of the present invention, each 10 μL PCR reaction system consists of the following components: 5.0 μL 2× TaqPCR Master Mix, 0.5 μL of upstream primer at a concentration of 10 pmol / μL, 0.5 μL of downstream primer at a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, 20 ng of DNA template. The upstream primer consists of two primers: an upstream primer with an adapter and an adapter primer with a fluorescent group.

[0015] As a preferred embodiment of the present invention, the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; 72℃ end extension for 20 min.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes SSR markers with high genetic recognition in black sea bream to analyze the genetic characteristics of black sea bream, thereby enabling the assessment of the genetic background of wild black sea bream populations before the implementation of stock enhancement and release, and providing a genetic basis for scientifically evaluating the release effect and formulating reasonable release strategies.

[0017] This invention further utilizes multiple combination designs and screenings to successfully construct nine-pair and ten-pair fluorescent PCR combinations. Because nine or ten primer pairs can be added simultaneously to the same reaction system, nine or ten target sites can be amplified at once, enabling genotypic identification at multiple sites. The PCR method established using this combination significantly improves detection efficiency, reduces detection costs, minimizes false positives and human error, and saves time to the greatest extent possible. It is the preferred method for evaluating the genetic effects of stock enhancement and release, identifying individuals / families, pedigree analysis, and evaluating germplasm resources.

[0018] Compared to conventional PCR, the fluorescent multiplex PCR constructed in this invention is a genetic invention for black sea bream, a method for evaluating the genetic effects of propagation and release, and a method for assessing germplasm resources, which has higher throughput, higher accuracy, higher efficiency, lower cost, and greater practical value. Attached Figure Description

[0019] Figures 1-9 This is a peak diagram of the genotyping detection using nine-fold fluorescent PCR; Figures 10-19 This is a peak diagram of the typing detection using a deca-fold fluorescent PCR assay. Figures 20-23 This is a peak diagram of genotyping detection in a nine-fold fluorescent PCR comparative example with four sites replaced in the comparative example; Figures 24-27 This is a peak diagram of genotyping detection in a nine-fold fluorescent PCR comparative example with three sites replaced in the comparative example; Figures 28-31This is a peak diagram of genotyping detection in a nine-fold fluorescent PCR comparative example where one site was replaced. Figures 32-35 This is a peak diagram of genotyping detection in a deca-fluorescence PCR comparative example with four sites replaced in the comparative example; Figures 36-39 This is a peak diagram of genotyping detection in a deca-symbol fluorescent PCR comparison with three sites replaced in the comparison example; Figures 40-43 This is a peak diagram of genotyping detection in a deca-symbol fluorescent PCR comparison sample with one site replaced. Figures 1-43 The horizontal axis represents the length of the DNA fragment, in bp, used to distinguish alleles of different sizes, with the peak position indicating the size of the allele fragment; the vertical axis represents the fluorescence intensity, in RFU, used to quantify the concentration of the DNA fragment, with the peak height, i.e., the vertical axis, representing the fluorescence signal intensity. Detailed Implementation

[0020] To illustrate the technical content, structural features, objectives, and effects of the technical solution in detail, the following description is provided in conjunction with specific embodiments and accompanying drawings. Unless otherwise specified, all experimental reagents used in the following embodiments are commercially available.

[0021] 1. Sample collection In the spring of 2024, 60 wild black seabream samples were collected from two sea areas awaiting release, namely the nearshore waters of Jianghong, Guangxi and Zhanjiang, Guangdong. After morphological identification, the dorsal muscle tissue was placed in a 95% alcohol solution and stored in a -20°C refrigerator for subsequent experiments.

[0022] 2. Genomic DNA extraction Genomic DNA was extracted from black sea bream using a magnetic bead-based tissue and blood genomic extraction kit. The integrity, purity, and concentration of the DNA were tested using 1% agarose gel electrophoresis and an ultra-micro biodetector. DNA that met the requirements for integrity, purity, and concentration was used for subsequent experiments.

[0023] 3. Synthesis of typing primers Based on the microsatellite sequences of the black sea bream that our team has preliminarily screened, we synthesized genotyping primers for 31 polymorphic sites using the adapter method. Specifically, a 21 bp adapter sequence was added to the upstream primer during synthesis. The sequence is: 5'-GAAGGTGACCAAGTTCATGCT-3', SEQ ID NO. 61. The sequences of the 31 microsatellite sites for the black sea bream are detailed in Table 1. Information on the 31 pairs of genotyping primers is detailed in Table 2.

[0024] Table 1. Sequences of 31 microsatellite loci in black sea bream.

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Table 2 Information on 31 pairs of typing primers

[0032] 4. Fluorescent PCR amplification DNA from six random Zhanjiang samples was amplified by gradient PCR at 52–62 °C using 31 pairs of genotyping primers and the adapter method to determine the optimal amplification conditions and polymorphism of each microsatellite locus for fluorescent PCR.

[0033] The total PCR reaction volume was 10 μL, including: 5.0 μL 2× Taq The PCR Master Mix consists of 0.5 μL of upstream primer at a concentration of 10 pmol / μL, 0.5 μL of downstream primer at a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, and 1.0 μL of DNA template containing approximately 20 ng. The upstream primers include two primers: an upstream primer with an adapter and an adapter primer with a fluorescent group.

[0034] The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; 72℃ end extension for 20 min.

[0035] 5. Fluorescent capillary electrophoresis detection Fluorescent capillary electrophoresis was used to perform typing detection on fluorescent PCR amplification products with high yield and high specificity.

[0036] The genotyping detection system consists of 10 μL, including 1.0 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500LIZ, and 8.5 μL of Hi-Di™ Formamide.

[0037] The test system was placed on a PCR instrument and denatured at 95°C for 3 minutes, then immediately cooled. Following the instrument operation procedure and SSR analysis detection program, 31 SSR samples were genotyped. GeneMarker software was used to analyze the genotyping results, obtaining the peak diagram, allele count, and genotype for each sample.

[0038] 6. Results of polymorphic primer screening Primer quality was evaluated based on polymorphic information content and fluorescence capillary electrophoresis band detection, and 27 loci and their primers were selected for subsequent population genotyping. The primer evaluation table for the 27 polymorphic microsatellite loci of the black sea bream is detailed in Table 3.

[0039] Table 3 Primer Evaluation Table

[0040] 7. Combination optimization of multiplex fluorescent PCR From 27 SSR loci with good polymorphism, 20 loci with similar primer annealing temperatures were selected. Based on allele intervals and amplification efficiency, four multiplex PCR combinations were formed, and the amplification system was optimized. To effectively reduce band slip, significantly increase polymorphism, minimize non-specific amplification, and improve amplification efficiency and genotyping accuracy, each multiplex PCR combination must include tri-, tetra-, penta-, and hexa-base microsatellite loci to construct a balanced and efficient multiplex PCR detection system. FAM, HEX, ROX, or TAMRA fluorescent groups were added to the 5' end of the upstream primer at each locus. Loci with the same fluorescent group must have different allele intervals.

[0041] The four multiplex PCR combinations are as follows: Combination 1: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, with a primer ratio of 1:1.5:2:2:1:2:3:5:7:7; FAM fluorescent groups are added to the 5' ends of the upstream primers of AS5-4, AS6-2, and AS6-6; HEX fluorescent groups are added to AS4-6, AS5-9, and AS6-8; ROX fluorescent groups are added to AS3-6 and AS4-4; and TAMRA fluorescent groups are added to AS3-15 and AS3-7.

[0042] Combination 2: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, with a corresponding primer ratio of 1:2:3:3:1:2:4:7:9:8; the addition of fluorescent groups is the same as in combination 1.

[0043] Combination 3: AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, AS6-3; the corresponding primer ratio is 1∶2∶1.5∶1.5∶2∶1∶3∶7∶5∶7; FAM fluorescent groups are added to the 5' ends of the upstream primers of AS3-3, AS5-8, and AS6-5; HEX fluorescent groups are added to AS3-14, AS4-7, and AS6-4; ROX fluorescent groups are added to AS5-10 and AS5-2; and TAMRA fluorescent groups are added to AS5-12 and AS6-3.

[0044] Combination 4: AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, AS6-3; the corresponding primer ratio is 1∶3∶2∶3∶4∶2∶2∶6∶6∶9; the addition of fluorescent groups is the same as in combination 3.

[0045] Multiplex fluorescent PCR amplification was performed on the genomic DNA of four black sea bream collected from Jianghong using upstream and downstream primers with fluorescent groups.

[0046] The total PCR reaction volume was 10 μL, including 5.0 μL of 2× Taq The PCR Master Mix consisted of 0.5 μL of upstream primer at a concentration of 10 pmol / μL, 0.5 μL of downstream primer at a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, and 1.0 μL of DNA template containing approximately 20 ng. The total volume of the 10 upstream primers and 10 downstream primers was 0.5 μL.

[0047] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; extension at 72℃ for 20 min.

[0048] The PCR products were genotyped and analyzed using the methods described above. Genotyping results showed that none of the four groups of multiplex fluorescent PCR products achieved complete peak elution, and the peaks at some sites were narrow, indicating that the combined sites were not fully amplified or had low amplification efficiency.

[0049] Based on the peak elution results of four multiplex PCR combinations, the primer ratios for each locus in the multiplex fluorescent PCR amplification system were adjusted. After adjustment, two sets of multiplex fluorescent PCR combinations were obtained, as follows: Combination 5: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, with a primer ratio of 1:2:1:5:1:2:5:7:9:9; the fluorescent group is the same as in combination 1 above.

[0050] Combination 6: AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, AS6-3; the corresponding primer ratio is 2∶3∶2∶4∶6∶2∶4∶6∶6∶6; the addition of fluorescent groups is the same as in combination 3.

[0051] Based on the adjusted two sets of multiplex fluorescent PCR combinations, genomic DNA from 30 black seabream samples collected from Zhanjiang and Jianghong was amplified by multiplex fluorescent PCR. The PCR amplification procedure and genotyping method were the same as above. The genotyping results of the 30 samples showed that compared with the first multiplex PCR combination, the peak sites and signals of this combination increased, but the AS6-2 site in combination 1 failed to produce a peak, and the peak shape of some sites was still relatively narrow.

[0052] Based on the genotyping results of 30 samples, the primer ratios for each locus in the two sets of multiplex fluorescent PCR were optimized again, and multiplex fluorescent PCR amplification was performed again on the genomic DNA of the 30 black sea bream. The PCR amplification procedure and genotyping detection method were the same as above. The optimized combination is as follows: Combination 7: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, with a primer ratio of 3:5:3:3:2:2:5:7:9:9; the fluorescent group is the same as in combination 1 above.

[0053] Combination 8: AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, AS6-3; the corresponding primer ratio is 1∶2∶1∶3∶2∶2∶2∶6∶8∶9; the addition of fluorescent groups is the same as in combination 3.

[0054] The genotyping results of 30 samples showed that in multiplex fluorescent PCR combination 1, all 9 sites successfully emitted peaks, and the genotyping spectrum was clear and had high resolution, which could accurately interpret the genotyping results. However, the AS6-2 site failed to emit a peak, so the AS6-2 site was removed. In multiplex fluorescent PCR combination 2, all 10 sites successfully emitted peaks, and the genotyping spectrum was clear and had high resolution, which could accurately interpret the genotyping results.

[0055] In summary, this invention successfully constructed two sets of multiplex fluorescent PCR combinations with high amplification efficiency and good peak quality. Both sets of multiplex fluorescent PCR combinations have numerous combination sites, all of which are polymorphic three- to six-base repeat microsatellites. The primer design is reasonable, with no non-specific amplification. After multiple optimizations, each PCR reaction can be performed in parallel without interference within the same system, effectively amplifying the target fragment. Details of the nine-fold and ten-fold fluorescent PCR amplification systems are shown in Table 4. The peak diagrams for nine-fold fluorescent PCR genotyping detection of the samples are shown below. Figures 1-9 The peak diagram for the deca-symbol fluorescence PCR genotyping detection is shown below. Figures 10-19 It should be noted that this invention only uses the genotyping peak diagram of one sample to illustrate the effect.

[0056] Table 4. Nine-fold and ten-fold fluorescent PCR amplification systems

[0057] Comparative Example 1. Nine-fold fluorescent PCR To validate the nine-fold fluorescent PCR combination, primer pairs 1-4 in the nine-fold PCR combination were replaced with other high-quality primer pairs. The specific replacement scheme is as follows:

[0058] (1) Four pairs of primers at four sites were randomly replaced. The replacement sites were AS3-6, AS3-7, AS4-6 and AS6-6.

[0059] (2) Replace the three pairs of primers at three sites randomly: 1. Replace the three pairs of primers at sites AS3-7, AS4-6 and AS6-6; 2. Replace the three pairs of primers at sites AS3-6, AS4-6 and AS6-6; 3. Replace the three pairs of primers at sites AS3-6, AS3-7 and AS6-6; 4. Replace the three pairs of primers at sites AS3-6, AS3-7 and AS4-6.

[0060] (3) Randomly replace one primer at one site, and the replacement site is AS3-6, AS3-7, AS4-6 or AS6-6.

[0061] Prepare the PCR amplification system according to Table 4, and perform PCR amplification and capillary electrophoresis as described above. The results showed that after primer replacement, some or all sites in the nine-fold fluorescent PCR could not be successfully amplified. Figures 20-31 .

[0062] 2. Decadal fluorescent PCR To validate the ten-fold fluorescent PCR combination, primer pairs 1-4 in the ten-fold combination were replaced with other high-quality primer pairs. The specific replacement scheme is as follows:

[0063] (1) Four pairs of primers at four sites were randomly replaced. The sites replaced were AS3-14, AS5-2, AS5-8 and AS6-3.

[0064] (2) Replace the three pairs of primers at three sites randomly: First, replace the three pairs of primers at sites AS5-2, AS5-8 and AS6-3; Second, replace the three pairs of primers at sites AS3-14, AS5-8 and AS6-3; Third, replace the three pairs of primers at sites AS3-14, AS5-2 and AS6-3; Fourth, replace the three pairs of primers at sites AS3-14, AS5-2 and AS5-8.

[0065] (3) Randomly replace one primer at one site, and the replacement site is AS3-14, AS5-2, AS5-8 or AS6-3.

[0066] Prepare the PCR amplification system according to Table 4, and perform PCR amplification and capillary electrophoresis as described above. Results showed that after primer replacement, some or all sites in the deca-fold fluorescent PCR could not be successfully amplified. (See Table 4 for details.) Figures 32-43 .

[0067] This invention successfully screened out balanced and efficient nine-fold and ten-fold fluorescent PCR combinations, which can provide efficient, accurate and low-cost detection methods for the genetic characteristic analysis of black sea bream, and can provide strong technical support for the construction of a comprehensive assessment system for the genetic risk of black sea bream breeding and release. It has important significance in both scientific theory and fishery production practice.

[0068] Experimental Example Genetic baseline characteristics of wild black sea bream populations prior to stock enhancement and release 1. Sample collection In the spring of 2024, prior to the release of new fish, 30 wild black seabream samples were collected from the coastal waters of Jianghong, Guangxi and Zhanjiang, Guangdong, where the release was planned. After morphological identification, the dorsal muscle tissue was placed in a 95% alcohol solution and stored at -20°C for subsequent experiments.

[0069] 2. Genomic DNA extraction Genomic DNA was extracted from black sea bream using a magnetic bead-based tissue and blood genomic extraction kit. The integrity, purity, and concentration of the DNA were tested using 1% agarose gel electrophoresis and an ultra-micro biodetector. DNA that met the requirements for integrity, purity, and concentration was used for subsequent experiments.

[0070] 3. Nine-fold and ten-fold fluorescent PCR amplification For each individual, nine-fold and ten-fold fluorescent PCR amplification systems were prepared according to Table 4. The PCR reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; 72℃ extension for 20 min.

[0071] 4. Fluorescent capillary electrophoresis detection Genotyping of multiplex fluorescent PCR products was performed using the method described above. The genotyping system consisted of 10 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500 LIZ, and 8.5 μL of Hi-Di™ Formamide. The system was placed on the PCR instrument and denatured at 95°C for 3 min, then immediately cooled. Following the instrument operation procedure and SSR analysis program, genotyping of 30 SSR samples was performed using a 96-channel fully automated ABI 3730xL genetic analyzer. The genotyping results were analyzed using GeneMarker software to obtain the alleles and genotypes for each sample.

[0072] 5. Analysis and results of the genetic background characteristics of wild populations of black sea bream The genotyping results were compiled and statistically analyzed, and the number of alleles for microsatellite markers was calculated using GenALEx 6.51. Na Effective number of alleles Ne Shannon Information Index I Apparent heterozygosity Ho and expected heterozygosity He ;Calculate polymorphic information content using Cervus 3.0.7 PIC GenePop 4.8.3 was used to calculate the fixed exponents at each point. F .

[0073] The genetic background characteristics of wild black sea bream populations are shown in Table 5. The genetic characteristics of 19 loci in the total population of the proposed release areas of Zhanjiang and Jianghong are also included. Na The range is 3–24, of which 12 sites are highly polymorphic, and 6 ≤ Na =6~24; 7 sites are moderately polymorphic, 3≤ Na ≤5. Ne The range was 1.229–16.057, of which 13 sites exhibited high diversity, with 3 < 16.057. Ne =3.001~16.057; 4 sites showed moderate diversity, 1.5 < Ne =1.880~2.908≤3; only AS5-10 and AS6-5 sites showed low diversity. Ne=1.229~1.312≤1.5. I The range was 0.426–2.962, with 9 sites exhibiting high diversity (1.5 < 0.426). I =1.518~2.962; 8 loci showed moderate diversity, 0.5 < I =0.826~1.455<1.5; only AS5-10 and AS6-5 loci showed low diversity. I =0.426 and 0.493 < 0.5. Ho The value ranged from 0.200 to 0.933, with 15 sites showing high heterozygosity (0.5 ≤ 0.933). Ho =0.5~0.933; 4 loci showed moderate heterozygosity, 0.2≤ Ho =0.2~0.433<0.5. He The range was 0.186–0.938, with 16 sites showing high heterozygosity (≤0.5). He =0.548~0.938; two loci showed moderate heterozygosity, 0.2≤ He =0.238~0.468<0.5; only site AS5-10 is of low heterozygosity. PIC The values ​​ranged from 0.180 to 0.934, with 16 sites exhibiting high polymorphism (0.5 < 0.934). PIC =0.523~0.934; 3 sites showed moderate or low polymorphism. PIC =0.180~0.407<0.5. In summary, except for AS5-10 and AS6-5, the remaining 17 loci showed high polymorphism in the total population of the proposed release area; the total population of Zhanjiang and Jianghong in the proposed release area showed a high level of genetic diversity.

[0074] Among the Zhanjiang population in the proposed release area, 19 microsatellite loci were observed. Na The range is 3 to 14, with a mean of 6.368; Ne The range was 1.23 to 8.491, with a mean of 4.236; I The values ​​ranged from 0.435 to 2.384, with a mean of 1.455. Ho The values ​​ranged from 0.2 to 0.933, with a mean of 0.638. He The values ​​ranged from 0.187 to 0.882, with a mean of 0.680. PIC The values ​​ranged from 0.181 to 0.872, with a mean of 0.646. F The values ​​ranged from -0.331 to 0.547, with a mean of 0.048. Among the river flood population in the proposed release area, 19 microsatellite loci... Na The range is 2 to 16, with a mean of 6.737. Ne The range was 1.226 to 12.162, with a mean of 4.017; IThe values ​​ranged from 0.389 to 2.636, with a mean of 1.417. Ho The values ​​ranged from 0.2 to 0.929, with a mean of 0.600. He The values ​​ranged from 0.184 to 0.918, with a mean of 0.654. PIC The values ​​ranged from 0.175 to 0.912, with a mean of 0.618. F The mean values ​​ranged from -0.328 to 0.615, with a mean of 0.059. The mean values ​​of the genetic parameters for both the Zhanjiang and Jianghong populations showed... Na Greater than 6, Ne Greater than 3 I Greater than 1.5 Ho Greater than 0.5 He Greater than 0.5 PIC A value greater than 0.5 indicates that the wild populations in the two proposed release areas of Zhanjiang and Jianghong have a high level of genetic diversity, that is, they have a rich genetic variation base, suggesting that the two local populations are in good health and have strong environmental adaptability potential, making them suitable as parent stock for release in these two areas.

[0075] Table 5. Genetic analysis of 19 microsatellite loci of black seabream in the proposed release area.

[0076] Note: N Number of individuals; Na Number of alleles; Ne : Effective number of alleles; Ho : Observe heterozygosity; He Expected heterozygosity; I Shannon Information Index; PIC : Polymorphic information content; F : Fixed index.

[0077] The method of this invention is used to investigate and analyze the genetic background characteristics of black sea bream stocking fry and their breeding parents, in order to screen out healthy parents and stocking fry that match the genetic background of the wild population and have high diversity, and to assess the potential genetic risks of stocking from the source. After the stocking is implemented, the method of this invention is used to continue to assess the impact of the released population on the genetic characteristics of the wild population, thereby achieving dynamic monitoring of the genetic risks of stocking.

[0078] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. The application of an SSR marker detection kit in the genetic characteristic analysis of black sea bream, characterized in that, The SSR mark is selected from any one or a combination of several of the following: AS5-4, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12, and AS6-3. The repeating unit of AS5-4 is TCTAT, and the number of repetitions is 6. The repeating unit of AS6-6 is CGATGG, and the number of repetitions is 5. The repeating unit in AS4-6 is TCTA, and the number of repetitions is 15. AS5-9 has a repeating unit of GTGTT and a repeat count of 6. The repeating unit in AS6-8 is AGGAGA, and the number of repetitions is 5. AS3-6 uses the TTG as its repeating unit and repeats 8 times. The repeating unit of AS4-4 is GAA, and the number of repetitions is 5. The repeating unit of AS3-15 is AGA, and the number of repetitions is 12. The repeating unit of AS3-7 is TCA, and the number of repetitions is 8. The repeating unit of AS3-3 is CTT, and the number of repetitions is 18. The repeating unit in AS5-8 is TAGAA, and the number of repetitions is 9. The repeating unit of AS6-5 is AGGGTT, and the number of repetitions is 5. The repeating unit of AS3-14 is AAT, and the number of repetitions is 8. The repeating unit of AS4-7 is TTCT, and the number of repetitions is 9. The repeating unit in AS6-4 is CCTGGT, and the number of repetitions is 5. The repeating unit of AS5-10 is TTGTT, and the number of repetitions is 5. AS5-2's repeating unit is AACAT, and the number of repetitions is 7; The repeating unit of AS5-12 is TTAGA, and the number of repetitions is 9; The repeating unit in AS6-3 is CAAGTA, and the number of repetitions is 5.

2. A primer pair, characterized in that, The primer pair is used to detect the SSR marker of claim 1, and the sequence of the primer pair is as follows: The forward primer sequence for detecting AS3-6 is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The forward primer sequence for detecting AS3-7 is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The forward primer sequence for detecting AS3-15 is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; The forward primer sequence for detecting AS4-4 is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8; The forward primer sequence for detecting AS4-6 is shown in SEQ ID NO.9, and the reverse primer sequence is shown in SEQ ID NO.10; The forward primer sequence for detecting AS5-4 is shown in SEQ ID NO.11, and the reverse primer sequence is shown in SEQ ID NO.12; The forward primer sequence for detecting AS5-9 is shown in SEQ ID NO.13, and the reverse primer sequence is shown in SEQ ID NO.14; The forward primer sequence for detecting AS6-6 is shown in SEQ ID NO.15, and the reverse primer sequence is shown in SEQ ID NO.

16. The forward primer sequence for detecting AS6-8 is shown in SEQ ID NO.17, and the reverse primer sequence is shown in SEQ ID NO.

18. The forward primer sequence for detecting AS3-3 is shown in SEQ ID NO.19, and the reverse primer sequence is shown in SEQ ID NO.20; The forward primer sequence for detecting AS3-14 is shown in SEQ ID NO.21, and the reverse primer sequence is shown in SEQ ID NO.22; The forward primer sequence for detecting AS4-7 is shown in SEQ ID NO.23, and the reverse primer sequence is shown in SEQ ID NO.24; The forward primer sequence for detecting AS5-2 is shown in SEQ ID NO.25, and the reverse primer sequence is shown in SEQ ID NO.26; The forward primer sequence for detecting AS5-8 is shown in SEQ ID NO.27, and the reverse primer sequence is shown in SEQ ID NO.28; The forward primer sequence for detecting AS5-10 is shown in SEQ ID NO.29, and the reverse primer sequence is shown in SEQ ID NO.30; The forward primer sequence for detecting AS5-12 is shown in SEQ ID NO.31, and the reverse primer sequence is shown in SEQ ID NO.32; The forward primer sequence for detecting AS6-3 is shown in SEQ ID NO.33, and the reverse primer sequence is shown in SEQ ID NO.34; The forward primer sequence for detecting AS6-4 is shown in SEQ ID NO.35, and the reverse primer sequence is shown in SEQ ID NO.36; The forward primer sequence for detecting AS6-5 is shown in SEQ ID NO.37, and the reverse primer sequence is shown in SEQ ID NO.

38.

3. The primer pair according to claim 2, characterized in that, The forward primers for amplifying AS3-6, AS4-4, AS5-2, and AS5-10 have a fluorescent label ROX at their 5' ends; The forward primers for amplifying AS3-7, AS3-15, AS5-12, and AS6-3 have a fluorescent label TAMRA at their 5' ends; The forward primers for amplifying AS4-6, AS5-9, AS6-8, AS3-14, AS4-7, and AS6-4 have a 5' end labeled with HEX fluorescently. The forward primers for amplifying AS5-4, AS6-6, AS3-3, AS5-8, and AS6-5 have a fluorescent label FAM at their 5' ends.

4. A kit for analyzing the genetic characteristics of black sea bream, characterized in that, The kit includes the primer pair as described in claim 2.

5. The application of the primer pair of claim 2 or the kit of claim 4 in the genetic characterization of black sea bream.

6. The application according to claim 5, characterized in that, The primer pairs or the kit described herein are used to conduct a genetic baseline survey of wild black sea bream populations prior to the release of aquatic organisms.

7. A method for analyzing the genetic characteristics of black sea bream, characterized in that, Includes the following steps: Extract whole-genome DNA from the sample to be tested; PCR amplification of whole-genome DNA was performed using the primer pair described in claim 2; The amplified products were subjected to capillary electrophoresis, and the genetic characteristics of the black sea bream were analyzed based on the electrophoresis results. The primer pairs are composed of amplification primers of AS3-6, AS3-7, AS3-15, AS5-4, AS4-4, AS4-6, AS5-9, AS6-6 and AS6-8, or are composed of amplification primers of AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12 and AS6-3.

8. The method for analyzing the genetic characteristics of black sea bream according to claim 7, characterized in that, The mixing volume ratio of the amplification primers for AS3-6, AS3-7, AS3-15, AS4-4, AS4-6, AS5-4, AS5-9, AS6-6 and AS6-8 is 5∶9∶9∶7∶3∶3∶2∶3∶2, and the forward and reverse primers are mixed in equal proportions. The mixing volume ratio of the amplification primers for AS3-3, AS3-14, AS4-7, AS5-2, AS5-8, AS5-10, AS5-12, AS6-3, AS6-4, and AS6-5 is 1:3:2:6:2:2:8:9:2, and the forward and reverse primers are mixed in equal proportions.

9. The method for analyzing the genetic characteristics of black sea bream according to claim 7, characterized in that, Each 10 μL PCR reaction system consists of the following components: 5.0 μL 2× Taq PCR Master Mix, 0.5 μL of upstream primer at a concentration of 10 pmol / μL, 0.5 μL of downstream primer at a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, 20 ng of DNA template. The upstream primer consists of two primers: an upstream primer with an adapter and an adapter primer with a fluorescent group.

10. The method for analyzing the genetic characteristics of black sea bream according to claim 7, characterized in that, The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, gradient annealing at 62~52℃ for 30 s, extension at 72℃ for 30 s, for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, extension at 72℃ for 30 s, for 25 cycles; and 72℃ end extension for 20 min.

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