Application of SSR marker detection kit in genetic characteristics analysis of black porgy

Abstract

The application belongs to the technical field of biological detection, and particularly relates to application of a SSR marker detection kit in genetic characteristic analysis of black porgy. 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 and AS5-12. The SSR marker is used to construct a nine-reagent fluorescent PCR combination and a ten-reagent fluorescent PCR combination, so that multi-site genotyping can be realized, the detection efficiency can be obviously improved, the detection cost can be reduced, false positive phenomena and human errors can be reduced, and time can be saved to the greatest extent. The kit is preferred for genetic effect evaluation of propagation and release, individual / family identification, family tracing analysis and evaluation of germplasm resources.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to application of a SSR marker detection kit in genetic characteristic analysis of black porgy, and is particularly used for genetic characteristic analysis of wild populations of black porgy before proliferation and release. BACKGROUND

[0002] Black porgy Acanthopagrus schlegelii Also known as black seabream, the black porgy is a nearshore warm-water bottom fish, which has the advantages of fast growth, wide food, salt tolerance, wide temperature, delicious meat, small moving range and strong disease resistance. In recent years, due to overfishing, habitat destruction and deterioration of fishery ecological environment, the wild resources of the black porgy have been seriously degraded, which affects the sustainability of fishery resources. In order to restore the resources of the black porgy and promote the sustainable use of fishery resources, the combination of natural proliferation of wild resources and release of artificially bred fry has become the main restoration measure. However, the current proliferation and release of the black porgy is based on extensive scale expansion, and the proliferation target is more focused on the increase of resource quantity, and the influence of the proliferation activity on the genetic characteristics of natural germplasm is lack of research, that is, the genetic risk assessment of the proliferation and release is ignored, which is not adaptive to the initial intention of fishery resource conservation and the requirement of sustainable fishery development goal.

[0003] A large number of studies have shown that there are obvious genetic differences between artificially bred fry and wild populations due to too few effective parent populations, too many generations of reproduction, different water areas or different populations, and a large-scale proliferation and release will destroy the gene pool of wild populations, affect the genome of wild individuals expressing local adaptability, change the genetic composition of the population, reduce the level of genetic variation, cause genetic homogenization, and further reduce the level of genetic differentiation, genetic fitness and health level, and cause a series of genetic risks.

[0004] In order to reduce the genetic risk of proliferation and maximize the proliferation benefit, some scholars believe that the genetic risk should be effectively prevented and evaluated from the following two aspects: first, the genetic risk caused by the proliferation release should be evaluated from the source, that is, before the implementation of the proliferation release, the genetic background characteristics of the wild population of the proliferation species, the proliferation seedlings and their breeding parents are investigated, so that the genetic background of the proliferation population is maximized to fit the wild population, so as to reduce the negative genetic influence of the proliferation release on the gene pool of the wild population; secondly, during the proliferation release process, the dynamic monitoring of the genetic risk of the proliferation release is carried out according to the influence of the release population on the genetic characteristics of the wild population, and is implemented throughout the proliferation release activity, so as to scientifically guide the proliferation release from the genetic point of view. As can be seen from the above, before and after the proliferation release, the effective prevention and evaluation of the genetic risk of the proliferation release is the inevitable requirement for the scientific and orderly development of the proliferation release and the effective management of the genetic resources. Therefore, before and after the proliferation release, it is necessary to analyze the genetic characteristics of the black plectrypops and evaluate the genetic risk of the proliferation release of the black plectrypops. SUMMARY

[0005] Therefore, the application uses the SSR marker with high genetic recognition in the black plectrypops to realize efficient genotyping of large-scale samples, so as to evaluate the genetic risk of the proliferation release of the black plectrypops.

[0006] The specific technical scheme provided by the application is as follows:

[0007] The application provides an application of an SSR marker detection kit in genetic characteristic analysis of black plectrypops, 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.

[0008] The repeat unit of AS5-4 is TCTAT, and the repeat number is 6.

[0009] The repeat unit of AS6-6 is CGATGG, and the repeat number is 5.

[0010] The repeat unit of AS4-6 is TCTA, and the repeat number is 15.

[0011] The repeat unit of AS5-9 is GTGTT, and the repeat number is 6.

[0012] The repeat unit of AS6-8 is AGGAGA, and the repeat number is 5.

[0013] The repeat unit of AS3-6 is TTG, and the repeat number is 8.

[0014] The repeat unit of AS4-4 is GAA, and the repeat number is 5;

[0015] The repeat unit of AS3-15 is AGA, and the repeat number is 12;

[0016] The repeat unit of AS3-7 is TCA, and the repeat number is 8;

[0017] The repeat unit of AS3-3 is CTT, and the repeat number is 18;

[0018] The repeat unit of AS5-8 is TAGAA, and the repeat number is 9;

[0019] The repeat unit of AS6-5 is AGGGTT, and the repeat number is 5;

[0020] The repeat unit of AS3-14 is AAT, and the repeat number is 8;

[0021] The repeat unit of AS4-7 is TTCT, and the repeat number is 9;

[0022] The repeat unit of AS6-4 is CCTGGT, and the repeat number is 5;

[0023] The repeat unit of AS5-10 is TTGTT, and the repeat number is 5;

[0024] The repeat unit of AS5-2 is AACAT, and the repeat number is 7;

[0025] The repeat unit of AS5-12 is TTAGA, and the repeat number is 9;

[0026] The repeat unit of AS6-3 is CAAGTA, and the repeat number is 5.

[0027] In a second aspect of the present application, a primer pair for detecting the SSR marker is provided, and the sequence of the primer pair is as follows:

[0028] 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;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] 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;

[0040] 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;

[0041] 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;

[0042] 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;

[0043] 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;

[0044] The forward primer sequence for detecting AS6-3 is shown as SEQ ID NO. 33, and the reverse primer sequence is shown as SEQ ID NO. 34;

[0045] The forward primer sequence for detecting AS6-4 is shown as SEQ ID NO. 35, and the reverse primer sequence is shown as SEQ ID NO. 36;

[0046] The forward primer sequence for detecting AS6-5 is shown as SEQ ID NO. 37, and the reverse primer sequence is shown as SEQ ID NO. 38.

[0047] As a preferred embodiment of the present application, the 5' end of the forward primer for amplifying AS3-6, AS4-4, AS5-2, and AS5-10 is labeled with fluorescent marker ROX;

[0048] The 5' end of the forward primer for amplifying AS3-7, AS3-15, AS5-12, and AS6-3 is labeled with fluorescent marker TAMRA;

[0049] The 5' end of the forward primer for amplifying AS4-6, AS5-9, AS6-8, AS3-14, AS4-7, and AS6-4 is labeled with fluorescent marker HEX;

[0050] The 5' end of the forward primer for amplifying AS5-4, AS6-6, AS3-3, AS5-8, and AS6-5 is labeled with fluorescent marker FAM.

[0051] In a third aspect of the present application, a kit for genetic characteristic analysis of black porgy is provided, which comprises the primer pair.

[0052] In a fourth aspect of the present application, the use of the SSR marker, the primer pair, or the kit in genetic characteristic analysis of black porgy is provided.

[0053] As a preferred embodiment of the present application, the SSR marker, the primer pair, or the kit is used for investigating the genetic background of wild population of black porgy before the implementation of proliferation and release.

[0054] In a fifth aspect of the present application, a method for genetic characteristic analysis of black porgy is provided, which comprises the following steps:

[0055] Extracting whole genome DNA of the sample to be tested;

[0056] Performing PCR amplification on the whole genome DNA by using the primer pair;

[0057] Performing capillary electrophoresis on the amplification product, and performing genetic characteristic analysis of black porgy according to the electrophoresis result;

[0058] The primer pair is combined by the amplification primers of AS3-6, AS3-7, AS3-15, AS5-4, AS4-4, AS4-6, AS5-9, AS6-6 and AS6-8, or is combined by the amplification primers of AS3-3, AS5-8, AS6-5, AS3-14, AS4-7, AS6-4, AS5-10, AS5-2, AS5-12 and AS6-3.

[0059] As a preferred embodiment of the present application, the mixed 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.

[0060] The mixed volume ratio of the amplification primers of 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.

[0061] As a preferred embodiment of the present application, 10 μL of PCR reaction system is composed of the following components: 5.0 μL of 2x PCR Master Mix, 0.5 μL of upstream primer with a concentration of 10 pmol / μL, 0.5 μL of downstream primer with a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, 20 ng of DNA template, the upstream primer including two primers: the upstream primer with a linker and the linker primer with a fluorescent group. Taq

[0062] As a preferred embodiment of the present application, the PCR amplification program is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62-52℃ gradient annealing for 30 s, 72℃ extension for 30 s, running for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, running for 25 cycles; 72℃ terminal extension for 20 min.

[0063] Compared with the prior art, the present application has the following beneficial effects:

[0064] The present application can analyze the genetic characteristics of black porgy by using the SSR marker with high genetic recognition in black porgy, so as to evaluate the genetic background of wild population of black porgy before the implementation of proliferation and release, and provide genetic basis for scientific evaluation of release effect and formulation of reasonable release strategy.

[0065] ​The application further successfully constructs a nine-fold fluorescence PCR combination and a ten-fold fluorescence PCR combination through multiple combination design and screening. Since 9 or 10 pairs of primers can be added in the same reaction system, 9 or 10 target sites can be amplified at one time, and genotype identification of multiple sites can be realized. The PCR method established by using the combination can obviously improve the detection efficiency, reduce the detection cost, reduce the false positive phenomenon and human error, and save time to the greatest extent, and is the first choice for genetic effect evaluation of proliferation release, individual / family identification, family tracing analysis and germplasm resource evaluation.

[0066] Compared with the conventional PCR, the fluorescence multiplex PCR constructed by the application is a genetic method of A. nigricans, genetic effect evaluation of proliferation release and germplasm resource evaluation, which has higher detection flux, higher accuracy, higher efficiency, lower cost and greater practical value. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figures 1-9 is a typing detection peak graph of the nine-fold fluorescence PCR;

[0068] Figures 10-19 is a typing detection peak graph of the ten-fold fluorescence PCR;

[0069] Figures 20-23 is a typing detection peak graph of the nine-fold fluorescence PCR in the comparative example of replacing 4 sites;

[0070] Figures 24-27 is a typing detection peak graph of the nine-fold fluorescence PCR in the comparative example of replacing 3 sites;

[0071] Figures 28-31 is a typing detection peak graph of the nine-fold fluorescence PCR in the comparative example of replacing 1 site;

[0072] Figures 32-35 is a typing detection peak graph of the ten-fold fluorescence PCR in the comparative example of replacing 4 sites;

[0073] Figures 36-39 is a typing detection peak graph of the ten-fold fluorescence PCR in the comparative example of replacing 3 sites;

[0074] Figures 40-43 is a typing detection peak graph of the ten-fold fluorescence PCR in the comparative example of replacing 1 site;

[0075] Figures 1-43 The horizontal coordinates in the above figures are all the length of DNA fragments, in units of bp, for distinguishing different sizes of alleles, and the peak position indicates the size of the allele fragment; the vertical coordinates are all the fluorescence intensity, in units of RFU, for quantifying the concentration of the DNA fragment, and the peak height, i.e. the vertical coordinate, indicates the fluorescence signal intensity. DETAILED DESCRIPTION

[0076] To make the technical content, structural features, purposes and effects of the technical solutions clear, specific examples are described in detail below in conjunction with the drawings. In the following examples, experimental reagents are obtained through commercial means unless otherwise specified.

[0077] 1. Sample collection

[0078] In spring 2024, 60 wild samples of black puffer were collected from two black puffer release sea areas, Jianghong in Guangxi and Zhanjiang in Guangdong. After morphological identification, the dorsal muscle tissue was placed in 95% alcohol solution and stored in a -20℃ refrigerator for subsequent experiments.

[0079] 2. Genomic DNA extraction

[0080] The genomic DNA of black puffer was extracted using the magnetic bead method and blood genomic extraction kit. The DNA integrity, purity and concentration were detected using 1% agarose gel electrophoresis and ultramicro biological detector. The DNA meeting the requirements of integrity, purity and concentration was used for subsequent experiments.

[0081] 3. Typing primer synthesis

[0082] Based on the microsatellite sequences of black puffer preliminarily screened by the team, 31 polymorphic site typing primers were synthesized using the linker method, i.e. a 21bp linker sequence was added to the upstream primer during synthesis, and the sequence was: 5'-GAAGGTGACCAAGTTCATGCT-3', SEQ ID NO. 61. The sequences of the 31 microsatellite sites of black puffer are shown in Table 1. The information of the 31 pairs of typing primers is shown in Table 2.

[0083] Table 1 Sequences of 31 microsatellite sites of black puffer

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Table 2 Information of 31 pairs of typing primers

[0092]

[0093] 4. Fluorescent PCR amplification

[0094] DNA of 6 samples from Zhanjiang was randomly selected, and 31 pairs of typing primers were used to perform gradient PCR amplification at 52-62°C by using the adapter method to determine the optimal amplification conditions and polymorphism of each microsatellite site.

[0095] The total PCR reaction system was 10 μL, including: 5.0 μL 2x Taq PCR Master Mix, 0.5 μL of upstream primer with a concentration of 10 pmol / μL, 0.5 μL of downstream primer with a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, 1.0 μL of DNA template containing about 20 ng, wherein the upstream primer includes two primers: an upstream primer with an adapter and an adapter primer with a fluorescent group.

[0096] The PCR amplification program was set as: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, gradient annealing at 62-52°C for 30 s, 72°C extension for 30 s, running for 10 cycles; 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, running for 25 cycles; 72°C terminal extension for 20 min.

[0097] 5. Fluorescent capillary electrophoresis detection

[0098] Fluorescent capillary electrophoresis was used to detect the typing of the fluorescent PCR amplification product with high yield and strong specificity.

[0099] The typing detection system was 10 μL, including 1.0 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500LIZ, and 8.5 μL of Hi-Di™ Formamide.

[0100] The detection system was placed on the PCR instrument, denatured at 95°C for 3 min, and immediately cooled. Referring to the on-machine operation process and SSR analysis detection program, 31 SSR samples were typed and detected. The typing results were analyzed using GeneMarker software to obtain the peak graph, allele number, and genotype of each sample.

[0101] 6. Polymorphic primer screening results

[0102] Based on the size of polymorphic information content and the fluorescent capillary electrophoresis detection band, the quality of the primers was evaluated, and 27 sites and their primers were selected for subsequent population typing. The primer evaluation table of 27 polymorphic microsatellite sites of Plectryagus vespilus is shown in Table 3.

[0103] Table 3 Primer evaluation table

[0104]

[0105] 7. Combination optimization of multiplex fluorescent PCR

[0106] From 27 polymorphic SSR loci, 20 loci with similar annealing temperature were selected, and according to the allele interval and amplification efficiency, four multiplex PCR combinations were combined and the amplification system was optimized. In order to effectively reduce the slip band phenomenon, significantly increase the polymorphism, maximize the reduction of non-specific amplification, improve the amplification efficiency and typing accuracy, each multiplex PCR combination needs to contain three-base, four-base, five-base and six-base microsatellite loci to construct a balanced and efficient multiplex PCR detection system; FAM, HEX, ROX or TAMRA fluorescent groups are added to the 5' end of the upstream primer of each locus, and the loci with the same fluorescent group need to have different allele intervals.

[0107] The four multiplex PCR combinations are as follows:

[0108] Combination 1: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, the corresponding primer ratio is 1:1.5:2:2:1:2:3:5:7:7; FAM fluorescent group is added to the 5' end of the upstream primer of AS5-4, AS6-2, AS6-6, HEX fluorescent group is added to AS4-6, AS5-9, AS6-8; ROX fluorescent group is added to AS3-6, AS4-4; TAMRA fluorescent group is added to AS3-15, AS3-7.

[0109] Combination 2: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, the corresponding primer ratio is 1:2:3:3:1:2:4:7:9:8; the addition of fluorescent groups is the same as combination 1.

[0110] 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 group is added to the 5' end of the upstream primer of AS3-3, AS5-8, AS6-5, HEX fluorescent group is added to AS3-14, AS4-7, AS6-4; ROX fluorescent group is added to AS5-10, AS5-2; TAMRA fluorescent group is added to AS5-12, AS6-3.

[0111] 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 combination 3.

[0112] The four genomic DNAs of Aethaloperca rogaa collected from river flood were subjected to multiplex fluorescent PCR amplification by using upstream primers and downstream primers with fluorescent groups.

[0113] The total PCR reaction system was 10 μL, including 5.0 μL 2× Taq PCR Master Mix, 0.5 μL upstream primer with a concentration of 10 pmol / μL, 0.5 μL downstream primer with a concentration of 10 pmol / μL, 3.0 μL sterilized ultrapure water, 1.0 μL DNA template containing about 20 ng, and the total volume of 10 upstream primers and 10 downstream primers was 0.5 μL.

[0114] The PCR amplification program was as follows: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 62-52 ℃ gradient annealing for 30 s, 72 ℃ extension for 30 s, running for 10 cycles; 95 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 30 s, running for 25 cycles; 72 ℃ extension for 20 min.

[0115] The PCR products were subjected to typing detection and result analysis according to the above method. The typing detection result showed that the four groups of multiplex fluorescent PCR products could not be completely peaked successfully, and the peak type of individual site was relatively narrow, indicating that the combination sites were not completely amplified successfully or the amplification efficiency was relatively low.

[0116] Based on the peaking results of the four multiplex PCR combinations, the primer ratio of each site in the multiplex fluorescent PCR amplification system was adjusted. Two groups of multiplex fluorescent PCR combinations were obtained after adjustment, as follows:

[0117] Combination 5: AS5-4, AS6-2, AS6-6, AS4-6, AS5-9, AS6-8, AS3-6, AS4-4, AS3-15, AS3-7, the corresponding primer ratio is 1:2:1:5:1:2:5:7:9:9; the fluorescent groups are the same as combination 1.

[0118] 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 combination 3.

[0119] 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.

[0120] 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:

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] Table 4 Nine-plex and ten-plex fluorescent PCR amplification system

[0126]

[0127] Comparative example

[0128] 1. Nine-plex fluorescent PCR

[0129] To verify the nine-plex fluorescent PCR combination, the first four pairs of amplification primers in the nine-plex PCR combination were replaced by other high-quality primer pairs in turn. The specific replacement scheme is as follows:

[0130] (1) Randomly replace four pairs of primers at four sites, and the replacement sites are AS3-6, AS3-7, AS4-6 and AS6-6.

[0131] (2) Randomly replace three pairs of primers at three sites: one, replace the three pairs of primers at sites AS3-7, AS4-6 and AS6-6; two, replace the three pairs of primers at sites AS3-6, AS4-6 and AS6-6; three, replace the three pairs of primers at sites AS3-6, AS3-7 and AS6-6; four, replace the three pairs of primers at sites AS3-6, AS3-7 and AS4-6.

[0132] (3) Randomly replace one pair of primers at one site, and the replacement site is AS3-6, AS3-7, AS4-6 or AS6-6.

[0133] Prepare the PCR amplification system according to Table 4, and perform PCR amplification and capillary electrophoresis according to the above method. The results show that after replacing the primers, part or all of the sites in the nine-plex fluorescent PCR cannot be successfully amplified, see Figures 20-31 .

[0134] 2. Ten-plex fluorescent PCR

[0135] To verify the ten-plex fluorescent PCR combination, the first four pairs of amplification primers in the ten-plex combination were replaced by other high-quality primer pairs in turn. The specific replacement scheme is as follows:

[0136] (1) Randomly replace four pairs of primers at four sites, and the replacement sites are AS3-14, AS5-2, AS5-8 and AS6-3.

[0137] (2) Randomly replace three pairs of primers at three sites: 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.

[0138] (3) Randomly replace the primer of 1 site, the replaced site is AS3-14, AS5-2, AS5-8 or AS6-3.

[0139] The PCR amplification system is prepared according to Table 4, and the PCR amplification and capillary electrophoresis are carried out according to the above method. The results show that, after replacing the primer, part or all of the sites in the ten-fold fluorescence PCR cannot be successfully amplified, see Figures 32-43 .

[0140] The present application successfully screens the balanced and efficient nine-fold fluorescence PCR combination and ten-fold fluorescence PCR combination, which can provide an efficient, accurate and low-cost detection method for the genetic characteristic analysis of black plectrypops, and can provide strong technical support for the construction of the genetic risk comprehensive evaluation system of black plectrypops propagation and release, which has important significance in scientific theory and fishery production practice.

[0141] Experimental example

[0142] Investigation of genetic background characteristics of wild populations of black plectrypops before propagation and release

[0143] 1. Sample collection

[0144] In the spring of 2024, i.e. before the propagation and release, 30 wild samples of black plectrypops were collected from Jianghong, Guangxi and Zhanjiang, Guangdong offshore. After morphological identification, the muscle tissue of their back was taken and placed in 95% alcohol solution and stored in a -20℃ refrigerator for subsequent experiments.

[0145] 2. Genomic DNA extraction

[0146] The genomic DNA of black plectrypops was extracted by using the magnetic bead method and blood genomic extraction kit. The DNA integrity, purity and concentration were detected by 1% agarose gel electrophoresis and ultramicro biological detector. The DNA with integrity, purity and concentration meeting the requirements was used for subsequent experiments.

[0147] 3. Nine-fold and ten-fold fluorescence PCR amplification

[0148] The nine-fold fluorescence PCR amplification system and the ten-fold fluorescence PCR amplification system were prepared according to Table 4 for each individual. The PCR reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62~52℃ gradient annealing for 30 s, 72℃ extension for 30 s, running for 10 cycles; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, running for 25 cycles; 72℃ extension for 20 min.

[0149] 4. Fluorescence capillary electrophoresis detection

[0150] 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.

[0151] 5. Analysis and results of the genetic background characteristics of wild populations of black sea bream

[0152] 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 .

[0153] 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.

[0154] 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; I The 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... Nagreater than 6, Ne greater than 3, I greater than 1.5, Ho greater than 0.5, He greater than 0.5, PIC greater than 0.5, indicating that the wild populations in Zhanjiang and Jianghong two areas of the proposed release have a high level of genetic diversity, that is, a rich genetic variation basis, suggesting that the two local populations are in good health, have strong environmental adaptation potential, and are suitable as the release parents in these two areas.

[0155] Table 5 Genetic analysis of 19 microsatellite loci of Sparus macrocephalus in the proposed release sea area

[0156]

[0157] Note: N : number of individuals; Na : number of alleles; Ne : number of effective alleles; Ho : observed heterozygosity; He : expected heterozygosity; I : Shannon's information index; PIC : polymorphic information content; F : fixation index.

[0158] The method of the present application is used to investigate and analyze the genetic background characteristics of the breeding of Sparus macrocephalus, and to screen out healthy parents and release fry with high genetic diversity and genetic background matching the wild population, and to evaluate the genetic risk that may be caused by the proliferation and release from the source. After the implementation of the proliferation and release, the method of the present application is used to evaluate the influence of the release population on the genetic characteristics of the wild population, so as to realize the dynamic monitoring of the genetic risk of the proliferation and release.

[0159] Although the above embodiments have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic creative concept, so the above description is only an embodiment of the present application, and does not limit the patent protection scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. The application of an SSR marker detection kit in the genetic characteristic analysis of black sea bream, characterized in that, The genetic characteristic analysis of the black sea bream was conducted by investigating the genetic baseline characteristics of the wild black sea bream population before the release program. The SSR markers were selected from one of the following two combinations: Combination 1: AS3-6, AS3-7, AS3-15, AS5-4, AS4-4, AS4-6, AS5-9, AS6-6 and AS6-8; Combination 2: 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. The primer pair used to detect the SSR marker has the following sequence: 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.

2. The application according to claim 1, 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.

3. 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 1; 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 genetic characteristics analysis of the black sea bream was a genetic baseline survey of the wild population of black sea bream before the release and stocking. 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.

4. The method for analyzing the genetic characteristics of black sea bream according to claim 3, 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.

5. The method for analyzing the genetic characteristics of black sea bream according to claim 4, 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 forward primer at a concentration of 10 pmol / μL, 0.5 μL of reverse primer at a concentration of 10 pmol / μL, 3.0 μL of sterile ultrapure water, 20 ng of DNA template. The forward primers consist of two primers: a forward primer with an adapter and an adapter primer with a fluorescent group.

6. The method for analyzing the genetic characteristics of black sea bream according to claim 4, 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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