A method for identifying mandarin fish germplasm in heilongjiang river basin

Abstract

This invention relates to a method for identifying the germplasm of *Culter alburnus* (a type of mandarin fish) in the Heilongjiang River Basin, belonging to the field of fish genetics and breeding. The method comprises: 1. Collecting parental population samples of *Culter alburnus* from the Heilongjiang River Basin; 2. Performing phenotypic data analysis on the collected samples; 3. Extracting DNA from the collected samples and analyzing the genotypes of parent individuals using a combination of six SSR marker primers to establish fingerprint pattern maps and genetic parameter ranges; 4. Combining phenotypic parameters, fingerprint pattern maps, and genetic parameters to identify the germplasm of *Culter alburnus* from the Heilongjiang River Basin. This invention develops specific microsatellite marker combinations from the *Culter alburnus* genome sequence and establishes a method for identifying parental *Culter alburnus* populations in the Heilongjiang River Basin by combining phenotypic and genetic characteristics. This method is conducive to the sustainable development and rational utilization of *Culter alburnus* populations in this unique habitat and promotes the healthy development of the *Culter alburnus* aquaculture industry in Northeast China.

Description

Technical Field

[0001] This invention belongs to the field of fish genetics and breeding, specifically relating to a method for identifying the germplasm of the mandarin fish in the Heilongjiang River Basin. Background Technology

[0002] The mandarin fish (Siniperca chuatsi) belongs to the order Perciformes, family Percichthyidae, and genus Siniperca. Its flesh is tender, delicious, nutritious, and boneless, making it a unique and valuable freshwater economic fish species in my country, and an important target for aquaculture. Mandarin fish are distributed in the Yangtze, Pearl, and Heilongjiang river basins, with an annual production of 530,000 tons. The main producing areas, Guangdong, Hubei, and Anhui, account for nearly 70% of the total. Currently, based on the southern populations of mandarin fish in the Yangtze River and other areas, and through systematic breeding and hybridization, seven new varieties have been approved by the state, with seedlings covering major aquaculture areas.

[0003] The mandarin fish (Ctenopharynx gracilistylus) of the Heilongjiang River basin, commonly known as "Ao Hua," ranks first among the "Three Flowers and Five Luos" of Northeast China and is an important aquaculture fish. Compared to the southern mandarin fish population, the Heilongjiang River mandarin fish breed has stronger resistance to adverse conditions (cold hardiness) and diseases, and can adapt to the long ice-covered winter. However, the cultivation of southern fry in Northeast China suffers from low survival rates and high overwintering mortality, becoming a bottleneck restricting the development of the mandarin fish aquaculture industry in the Heilongjiang River basin. Therefore, the rational development of the Heilongjiang River mandarin fish breed is imperative. The prerequisite for this is to identify the collected breed to eliminate losses caused by mixed breeds. (Invention Content)

[0004] This invention provides a method for identifying the germplasm of mandarin fish in the Heilongjiang River Basin.

[0005] The method for identifying the germplasm of Mandarin fish in the Heilongjiang River Basin according to the present invention is carried out according to the following steps:

[0006] I. Collect parental samples of Mandarin fish from the Heilongjiang River basin;

[0007] II. Perform phenotypic data analysis on the collected samples;

[0008] III. Extract DNA from the collected samples and analyze the genotypes of the parent individuals using primer combinations with six SSR markers to establish fingerprint patterns and genetic parameter ranges; the names of the SSR markers, primer sequences, and amplification combinations are as follows:

[0009] Multiplex PCR group I:

[0010] HLJ GY222

[0011] Upstream primer: ROX-5'-GATCA CCTAA ACCCA GACAC C-3'

[0012] Downstream primer: 5'-ATTTC CACCA TGATG CCAGC-3'

[0013] HLJGY134

[0014] Upstream primer: FAM-5'-GATAA GATGC CGTCA TGTGC-3'

[0015] Downstream primer: 5'-TCAAA CATAG GGCTC AACTG G-3'

[0016] HLJGY213

[0017] Upstream primer: HEX-5'-ACTTA CTGAC AGAAG CGTGC-3'

[0018] Downstream primer: 5'-CTGAT AGTGA CGTCA TCGCC-3'

[0019] HLJGY064

[0020] Upstream primers: TAMRA-5'-AGGTG ACGCA CAATT AGACC-3'

[0021] Downstream primer: 5'-GATAC ACTGC AGCCC ATAGG-3'

[0022] Multiplex PCR group II:

[0023] HLJGY122

[0024] Upstream primer: ROX-5'-ATCAC CCAAC TGCAG ATTCC-3'

[0025] Downstream primer: 5'-TCCAC AGCTA AGTTA GTCGC-3'

[0026] HLJGY112

[0027] Upstream primers: FAM-5'-TCTTT ACCTG GAGAG GACGG-3'

[0028] Downstream primer: 5'-GGCAT GTTAT GGTCA GGAGG-3'

[0029] HLJGY202

[0030] Upstream primer: HEX-5'-AAGTC TCGTT GTGGT GATGG-3'

[0031] Downstream primer: 5'-TCAGG AGGAG CAAAC AAAGG-3'

[0032] HLJGY033

[0033] Upstream primer: TAMRA-5'-TGTGA TGAGA TGCCT TGTCG-3'

[0034] Downstream primer: 5'-ATATA CACCA CTGCC ACAGG-3'

[0035] Multiplex PCR group III:

[0036] HLJGY043

[0037] Upstream primers: ROX-5'-TTTCT GGGCT GGGAT TTAGG-3'

[0038] Downstream primer: 5'-TATGC TCTAC CATGC ACTCG-3'

[0039] HLJGY182

[0040] Upstream primer: FAM-5'-GGCTC GCTAT TGAAC ATGG-3'

[0041] Downstream primer: 5'-GTTGA AGAGG TGGTT CCAGG-3'

[0042] HLJGY084

[0043] Upstream primer: HEX-5'-CAACG TAGGT ATTGC ACTGC-3'

[0044] Downstream primer: 5'-AATCG GCATG TCTCT TCTGC-3'

[0045] HLJGY163

[0046] Upstream primer: TAMRA-5'-TGGTC ACTTA CACAC AGTGG-3'

[0047] Downstream primer: 5'-TCTGA AATGG GCCAT TCTGC-3'

[0048] Multiplex PCR group IV:

[0049] HLJ GY241

[0050] Upstream primer: ROX-5'-CAAGT AGTGC CACCA AGAGG-3'

[0051] Downstream primer: 5'-TCGAC TCTGA ACAAT AGCTG C-3'

[0052] HLJGY016

[0053] Upstream primer: FAM-5'-TATGA AGGCC TATGT GCACG-3'

[0054] Downstream primer: 5'-GCGAT GTTGT GTTAC TCTCG-3'

[0055] HLJGY174

[0056] Upstream primer: HEX-5'-ATGCT TTCTG TGTGA CCTCC-3'

[0057] Downstream primer: 5'-CGGAT GCACA CTTAT AAACA GC-3'

[0058] HLJGY105

[0059] Upstream primer: TAMRA-5'-TTCTT GCTCT CACAC ACTCC-3'

[0060] Downstream primer: 5'-AGATA GATAG ATGGA CGGAC G-3'

[0061] Multiplex PCR group V:

[0062] HLJGY151

[0063] Upstream primer: ROX-5'-GACCT ACAGT AGACC TCAAG C-3'

[0064] Downstream primer: 5'-GCTGT AGCAC TGCAA TTTCC-3'

[0065] HLJGY074

[0066] Upstream primers: FAM-5'-TGGAT GAACC TTGCT TTAGG G-3'

[0067] Downstream primer: 5'-TCCCT TTGCA GAAGA ATCGC-3'

[0068] HLJGY054

[0069] Upstream primer: HEX-5'-AGTAG AGACA ACAGC AACGG-3'

[0070] Downstream primer: 5'-TCATG CTGTA ACTCT GCTCG-3'

[0071] HLJGY021

[0072] Upstream primer: TAMRA-5'-AAGCT GGAGA GATGT TTGGG-3'

[0073] Downstream primer: 5'-AGGAG GAAGG GTACT ACTTG G-3'

[0074] Multiplex PCR VI:

[0075] HLJ GY231

[0076] Upstream primer: ROX-5'-TGGAC AGCAG TGTAT GTTGG-3'

[0077] Downstream primer: 5'-CAAGG GTGTT CAAGG GTAGG-3'

[0078] HLJGY143

[0079] Upstream primers: FAM-5'-TCATC TGCAG GTCTG TTTGG-3'

[0080] Downstream primer: 5'-TGCAG ACTGG CACAA TTCC-3'

[0081] HLJGY095

[0082] Upstream primer: HEX-5'-GACTT CACAG AAGCA AAGCC-3'

[0083] Downstream primer: 5'-CCAAG CTTTG TTCAC ACAGC-3'

[0084] HLJGY192

[0085] Upstream primer: TAMRA-5'-GATAT GAGGC AACAC ACAGC-3'

[0086] Downstream primer: 5'-ACCCT CACAC TTTGT CTTAA CC-3';

[0087] IV. Identify the germplasm of Heilongjiang mandarin fish by combining phenotypic parameters, fingerprint pattern maps, and genetic parameters.

[0088] Furthermore, the phenotypic data in step two include the individual measurements of the mandarin fish's weight, total length, body length, body height, head length, snout length, caudal peduncle height, and caudal peduncle length.

[0089] Furthermore, the SSR marker combinations used to identify the genetic characteristics of mandarin fish germplasm in the Heilongjiang River Basin were derived from 24 chromosomes in the genome. Based on the size of the amplified fragments and amplification conditions, six multiplex PCR combinations were formed, and red-ROX, blue-FAM, green-HEX, or black-TAMRA fluorescence were labeled at the 5' end of the forward primer, respectively.

[0090] Furthermore, after the SSR-labeled amplification of genomic DNA reaction was completed, the reaction products were separated by capillary gel electrophoresis (3730XL genetic analyzer). Using LIZ500-orange as the molecular weight standard, red, blue, green, and black fluorescence were switched to collect images and data (GeneMappre V4.1 software) to obtain genotype data.

[0091] Furthermore, IBM SPSS Statistics 26.0 was used to statistically analyze the phenotypic parameters of the parents. The weight of the 3-year-old parent fish ranged from 1010.00 to 3690.00 g, with an average of (2061.85±689.03) g, and the body length ranged from 348.20 to 567.70 mm, with an average of (450.24±55.00) mm. mm, body shape characteristics: body length to body height ratio 2.66–2.99, average 2.82±0.07; body length to head length ratio 2.75–3.60, average 3.05±0.22; body length to caudal peduncle length ratio 6.32–10.36, average 8.18±0.92; body length to caudal peduncle height ratio 8.18–10.90, average 9.32±0.65; head length to snout length ratio 3.75–5.05, average 4.28±0.32.

[0092] Furthermore, fingerprint pattern maps were drawn using R language based on the genotypes of individual mandarin fish broodstock, and individuals containing the corresponding allele fragments originated from the Heilongjiang River basin.

[0093] Furthermore, the genetic analysis software used to analyze the parental genetic parameters was PopGene32, with 4–22 alleles, observed heterozygosity of 0.3889–0.9630, expected heterozygosity of 0.5626–0.9384, and polymorphism information content of 0.4897–0.9275.

[0094] The advantages and beneficial effects of this application are as follows:

[0095] 1. Multidimensional integrated discrimination with high accuracy. This invention breaks through the limitations of traditional single morphology or molecular markers, and for the first time organically combines phenotypic data, SSR fingerprints, and genetic parameters to comprehensively discriminate germplasm from three dimensions: appearance traits, DNA molecular level, and population genetic structure, significantly improving the accuracy and reliability of identification.

[0096] 2. The marker system is efficient and comprehensive. The 24 SSR markers screened using the method in this application are specifically distributed on different chromosomes of the mandarin fish genome. By optimizing their combination, six multiplex PCR amplification systems were constructed, enabling high-throughput and low-cost genotyping analysis and significantly improving detection efficiency.

[0097] 3. Fluorescent labeling technology enables automated and standardized genotyping. All forward primers are labeled with different colors of fluorescence (ROX, FAM, HEX, TAMRA). Combined with capillary electrophoresis and LIZ500 internal standard, products from multiple sites can be separated simultaneously. Software automatically collects images and data, avoiding human error in traditional electrophoresis, resulting in objective and reproducible results.

[0098] 4. The operating procedures are systematic and easy to promote. This application forms a complete and standardized operating procedure from sample collection, phenotypic measurement, DNA extraction, multiplex PCR amplification, capillary electrophoresis to data analysis, which is convenient for promotion and application in practical work such as germplasm identification, seedling management and resource protection.

[0099] 5. Specific germplasm identification criteria for mandarin fish in the Heilongjiang River Basin were established. This application obtained the phenotypic parameter ranges (such as body weight, body length, and body proportions), fingerprint pattern maps (specific allele combinations), and genetic parameter ranges (number of alleles 4–22, observed heterozygosity 0.3889–0.9630, polymorphism information content 0.4897–0.9275) of mandarin fish in this basin through large-scale parental population sampling, providing a clear and comparable control benchmark for subsequent identification.

[0100] 6. Effectively distinguishes different geographical groups and prevents genetic mixing. The discrimination method proposed in this application can accurately identify the native mandarin fish of the Heilongjiang River Basin, effectively distinguishing it from other river basins or artificially bred groups. This is of great significance for protecting local unique wild germplasm resources and avoiding genetic pollution.

[0101] 7. Provides scientific basis for germplasm resource evaluation and breeding. The obtained highly polymorphic SSR markers and genetic parameters (such as heterozygosity and polymorphic information content) can objectively reflect the level of genetic diversity in the population, providing key data support for germplasm resource assessment, parental selection and genetic management of Mandarin fish.

[0102] 8. Simple operation, controllable cost, and suitable for large-scale application. Multiplex PCR combination design reduces the number of reactions and reagent consumption. Fluorescent labeling combined with capillary electrophoresis enables rapid detection of batch samples, balancing accuracy, economy, and high efficiency, which is conducive to large-scale promotion in fisheries management departments, breeding enterprises, and research institutions.

[0103] 9. This invention fills a technological gap in the field of germplasm identification for mandarin fish in the Heilongjiang River Basin. Currently, there is no comprehensive molecular-morphological identification method specifically for mandarin fish in this basin. This invention fills this technological gap and provides an important technical tool for the protection of germplasm resources of cold-region fish.

[0104] This invention constructs a ternary comprehensive discrimination model integrating phenotypic data, SSR fingerprinting, and genetic parameters. By collecting parental samples of mandarin fish from the Heilongjiang River basin, measuring multiple morphological indicators such as body weight, total length, and body height, and utilizing a six-group multiplex PCR fluorescence amplification system composed of 24 specific SSR markers for efficient genotyping, combined with capillary electrophoresis and automated software analysis, accurate allele data is obtained. This establishes the unique phenotypic parameter range, fingerprint pattern map, and genetic parameter range for mandarin fish from this basin. This method overcomes the limitations of traditional single morphological or molecular marker methods, possessing outstanding advantages such as high throughput, standardization, strong objectivity, good reproducibility, and controllable cost. It can effectively distinguish native mandarin fish from the Heilongjiang River basin from other geographical groups, preventing germplasm mixing and genetic contamination. It also provides a scientific basis for germplasm resource evaluation, parental breeding, and genetic management of this species, filling a technological gap in the field of germplasm identification for cold-region endemic fish. It is suitable for large-scale application in fisheries management, breeding enterprises, and research institutions.

[0105] The technical solution described in this application effectively solves the problem of "germstone identification," that is, determining whether an unknown individual belongs to the germplasm resources of a specific geographical group in the Heilongjiang River Basin. This application constructs a three-dimensional comprehensive identification model: (1) Phenotypic data: measuring multiple morphological indicators such as body weight, total length, body height, and head-to-snout length ratio, and establishing the phenotypic parameter range unique to this group; (2) 24 SSR markers: covering 24 chromosomes in the genome, forming 6 sets of multiplex PCR fluorescence amplification systems, and establishing fingerprint pattern maps; (3) Genetic parameters: statistically analyzing the range of population genetic parameters such as the number of alleles (4~22), observed heterozygosity (0.3889~0.9630), and polymorphism information content (0.4897~0.9275). These phenotypic data and population genetic parameters are the core basis for identifying the germplasm source, which is not mentioned at all in the comparative patent. Moreover, the application goal of this application is to prevent the mixing of germplasm from different river basins—especially the inflow of southern seedlings into the north causing aquaculture losses, and to protect the unique wild germplasm resources of the Heilongjiang River Basin. Attached Figure Description

[0106] Figure 1 Phenotypic identification of mandarin fish germplasm in the Heilongjiang River Basin;

[0107] Figure 2 A pattern diagram of the DNA fingerprint of four microsatellite markers in Multiplex PCR group I of Mandarin fish from the Heilongjiang River Basin;

[0108] Figure 3 A pattern diagram of the DNA fingerprint of four microsatellite markers in Multiplex PCR group II of mandarin fish from the Heilongjiang River Basin;

[0109] Figure 4 A pattern diagram of the DNA fingerprint of four microsatellite markers in Multiplex PCR group III of Mandarin fish from the Heilongjiang River Basin;

[0110] Figure 5 A pattern diagram of the DNA fingerprint of four microsatellite markers in multiplex PCR group IV of mandarin fish from the Heilongjiang River Basin;

[0111] Figure 6 A pattern diagram of the DNA fingerprint of four microsatellite markers in Multiplex PCR group V of mandarin fish from the Heilongjiang River Basin;

[0112] Figure 7 A pattern diagram of the DNA fingerprint of four microsatellite markers from the Multiplex PCR group VI of the Mandarin fish in the Heilongjiang River Basin. Detailed Implementation

[0113] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0114] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0115] Example 1: Screening of novel microsatellite markers for mandarin fish

[0116] 1. The complete genome sequence of *Siniperca chuatsi* (GCF020085105.1) was downloaded from the GenBank database. The genome size was 718.8 Mb. Gene sequences from all 24 chromosomes were downloaded. Using Krait software (version 1.5.1), perfect microsatellite loci with tetranucleotide repeats in each chromosome sequence were identified, with a standard of ≥4 repeats. The flanking length of the retrieved SSR loci was 150 bp, and other parameters were set to default. Based on the location information, microsatellite primers with tetranucleotide repeats were designed using the Primer3 function in Krait. Six primer pairs were selected for each chromosome, resulting in a total of 144 primer pairs.

[0117] 2. Wild mandarin fish (Siniperca chuatsi) were collected from the Fuyuan section of the Heilongjiang River and the Hutou section of the Wusuli River and bred into parent fish populations. Aquaculture populations from the Songhua River (Nashui Agricultural Development Co., Ltd., Zhaodong City, Heilongjiang Province) and the Pearl River (Shunhuayuan Aquatic Products Co., Ltd., Sanshui District, Foshan City, Guangdong Province) were collected to form candidate populations. Two samples were randomly selected from each population, and genomic DNA was extracted as templates. A 15 μL PCR amplification reaction system was established, containing 2 μL template DNA (50 ng / μL), 11 μL PCR mixing buffer (final MgCl2 concentration 1.5 mmol / L, final dNTP concentration 200 μmol / L), 1 μL upstream and downstream primers (10 mmol / L), 0.2 μL Taq DNA polymerase (5 U / μL), and 0.8 μL sterile deionized water. The PCR program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 56–58 °C annealing and extension for 30 s, 72 °C extension for 30 s, for 27 cycles; final extension at 72 °C for 7 min. Bands were separated by 8% polyacrylamide gel electrophoresis to screen for markers with stable amplification, clear bands, and polymorphism, thus initially identifying markers showing inter-population differences.

[0118] 3. Results: Of the 144 primer pairs, 107 pairs amplified stable, clear, and polymorphic DNA bands with 2-8 alleles. Based on chromosome distribution, markers with more than 4 alleles and significant inter-population differences were selected. Multiplex PCR combinations were constructed, taking into account annealing temperature and fragment size. After preliminary amplification and verification, the next step of the experiment was conducted.

[0119] Example 2: Using the method of the present invention to determine the quality of mandarin fish collected in the Fuyuan section of the Heilongjiang and Wusuli Rivers.

[0120] 1. Two hundred live broodstock or reserve mandarin fish were collected from the Fuyuan section of the Heilongjiang River and the Hutou section of the Wusuli River. Preliminary identification was performed according to the external morphology specified in the People's Republic of China aquatic industry standard "Mandarin Fish" (SC 1038-2000). The fish were preserved at the Hulan Experimental Farm of the Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences. Fifty-four fish were sampled, and eight indicators (body weight, total length, body length, body depth, head length, snout length, caudal peduncle depth, and caudal peduncle length) were measured for each selected broodstock according to the People's Republic of China National Standard "Fish Germplasm Inspection" Part 3: Trait Determination (GB / T 18654.3-2026). The results showed that the average body weight of the broodstock was (2061.85±689.03) g, the average body length was (450.24±55.00) mm, and the average body depth was (159.67±19.76) mm. The body height (r=0.864) and body length (r=0.838) were the most correlated with body weight, while the body length-to-body height ratio was 2.66–2.99, with an average of 2.82 ± 0.07; the body length-to-head length ratio was 2.75–3.60, with an average of 3.05 ± 0.22; the body length-to-tail peduncle ratio was 6.32–10.36, with an average of 8.18 ± 0.92; the body length-to-tail peduncle-to-height ratio was 8.18–10.90, with an average of 9.32 ± 0.65; and the head length-to-snout length ratio was 3.75–5.05, with an average of 4.28 ± 0.32. Figure 1 (Phenological identification of mandarin fish germplasm in the Heilongjiang River Basin). Specific measurement data and statistical results are shown in Table 1 (Table 1: Phenotypic characteristics of parental mandarin fish populations in the Heilongjiang River Basin). Pit electronic markers (Yuya Technology (Shanghai) Co., Ltd.) were implanted into the left dorsal muscle of each individual, and the marker numbers were recorded to establish electronic phenotypic profiles for the parents. Simultaneously, a portion of fin tissue was cut, numbered, and dried on filter paper for future extraction of genomic DNA.

[0121] Table 1. Phenotypic characteristics of parental populations of Mandarin fish in the Heilongjiang River Basin

[0122] Body weight (g) 2061.85±689.03 1010.00 3690.00 0.704 −0.348 0.334 1.000 Total length (mm) 504.55±62.41 394.10 644.50 0.343 −0.734 0.124 0.831 Body length (mm) 450.24±55.00 348.20 567.70 0.337 −0.691 0.122 0.838 Body height (mm) 159.67±19.76 125.80 203.20 0.562 −0.467 0.124 0.864 Head length (mm) 147.89±19.52 110.60 203.80 0.712 0.607 0.132 0.760 snout length (mm) 34.62±5.26 24.10 52.00 0.488 1.374 0.152 0.582 Tailstock height (mm) 48.56±6.97 35.00 69.40 0.461 0.227 0.144 0.767 Tail shank length (mm) 55.26±8.72 36.70 77.40 0.182 −0.208 0.158 0.408 Body length to height ratio 2.82±0.07 2.66 2.99 0.255 −0.251 0.025 −0.126 Body length to head length ratio 3.05±0.22 2.75 3.60 0.786 −0.312 0.072 0.064 Body length to caudal peduncle length ratio 8.18±0.92 6.32 10.36 0.193 −0.333 0.112 0.278 Body length to caudal peduncle height ratio 9.32±0.65 8.18 10.90 0.445 0.035 0.070 −0.109 Head length to snout length ratio 4.28±0.32 3.75 5.05 0.370 −0.623 0.075 0.148

[0123] 2. Genomic DNA was extracted from the fin tissue of *Siniperca chuatsi* using a tissue genomic DNA extraction kit (Tiangen Biotech). After quantification using a UV spectrophotometer, the DNA was diluted to 50 ng / µL. Red (ROX), blue (FAM), green (HEX), or black (TAMRA) fluorescence was added to the 5' end of 24 polymorphic SSR marker forward primers. The genotypes of 96 samples were detected using capillary gel electrophoresis. Six multiplex PCR combinations were constructed using the 24 specific polymorphic primers. Primer information and amplification conditions are shown in Table 2 (Table 2: Specific SSR Primer Combinations Used for Genetic Monitoring of *Siniperca chuatsi* in the Heilongjiang River Basin). A 25 µL PCR reaction system was established, comprising 12.5 µL of 2×PCR mixing buffer (Thermo Fisher Scientific), 4 µL of primers (10 mmol / L) (0.5 µL each of the forward and reverse primers for each primer pair), 2 µL of DNA template (50 ng / µL), and 6.5 µL of sterile deionized water. The reaction program was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, annealing at 30 s, extension at 72°C for 30 s, for 25 cycles; and a final extension at 72°C for 5 min. After the reaction, the PCR product was diluted 10-fold, and 1 μL of the diluted PCR product was mixed with 10 μL of Hi-Di... TM Deionized formamide and 0.1 μL of ROX-500 molecular weight internal standard were used to prepare an electrophoresis mixture. The mixture was denatured at 95℃ for 5 min and immediately incubated on ice for 5 min. Capillary gel electrophoresis was performed on a 3730XL genetic analyzer (ABI). After electrophoresis, blue, green, black, and red fluorescence were switched, and images and data of the four groups of markers were collected using GeneMap-per V4.1 software. Figures 2-7 DNA fingerprint pattern for germplasm identification of mandarin fish in the Heilongjiang River Basin, among which Figure 2 A schematic diagram of the DNA fingerprinting pattern of four microsatellite markers in Multiplex PCR group I. Figure 3 A schematic diagram of the DNA fingerprinting pattern of four microsatellite markers in Multiplex PCR group II. Figure 4 A schematic diagram of the DNA fingerprinting pattern of four microsatellite markers in Multiplex PCR group III. Figure 5 A schematic diagram of the DNA fingerprinting pattern of four microsatellite markers in Multiplex PCR group IV. Figure 6 A schematic diagram of the DNA fingerprinting pattern of four microsatellite markers in Multiplex PCR group V. Figure 7DNA fingerprinting patterns of four microsatellite markers in Multiplex PCR group VI were generated using R language based on the genotypes of broodstock Mandarin fish. Individuals containing the corresponding allele fragments originated from the Heilongjiang River basin.

[0124] Table 2. Information on specific SSR primer combinations used in the genetic monitoring of mandarin fish germplasm in the Heilongjiang River Basin.

[0125] 3. Data conversion was performed using the software "Fish Germplasm Resource Genetic Analysis Device (ZL200710144749.3)". PopGene32 (Version 3.2) software was used to calculate the allele frequency (P) and allele count (N) for each microsatellite marker. o ), observed heterozygosity (H) o ), expected heterozygosity (H e ) and polymorphism information content (PIC). The resulting amplified fragment sizes ranged from 118 to 342 bp, and the number of alleles per marker (N) o The observed heterozygosity (H) ranges from 4 to 22. o The expected heterozygosity (H) ranged from 0.3889 to 0.9630. e The values ​​ranged from 0.5626 to 0.9384; the polymorphic information content (PIC) ranged from 0.4897 to 0.9275. Specific statistical data are shown in Table 3 (Table 3: Genetic Discriminant Parameters of Mandarin Fish Parents in the Heilongjiang River Basin).

[0126] Table 3. Genetic discrimination parameters of mandarin fish broodstock populations in the Heilongjiang River Basin

[0127]

[0128] Table 3 shows that the 24 SSR marker loci used in this invention all exhibited good polymorphism and high genetic information content in the broodstock population of *Mandarin fish* in the Heilongjiang River basin. Regarding fragment length, the amplification products of each marker ranged in size from 118 to 342 bp, with a wide distribution and little or no overlap within each multiplex PCR combination, ensuring that the six multiplex fluorescent amplification systems could perform efficient and accurate genotyping. In terms of allele count, the 24 loci had 4 to 22 alleles, with 17 loci having more than 10 alleles. The HLJGY174 locus had the most with 22 alleles, while the HLJGY202 locus had the fewest with 4. This rich allele diversity provided ample genetic marker resources for accurate differentiation between individuals. Regarding heterozygosity, the observed heterozygosity ranged from 0.3333 to 0.9630, while the expected heterozygosity ranged from 0.5626 to 0.9384. HLJGY021 had the highest observed heterozygosity at 0.9630, while HLJGY143 had the lowest at 0.3333 but still achieved an expected heterozygosity of 0.5626. The observed heterozygosity of most loci was close to the expected heterozygosity, indicating that the population was in good genetic equilibrium and the selected markers accurately reflected the population's genetic structure. Regarding polymorphism information content, the PIC values ​​of the 24 loci ranged from 0.4897 to 0.9275, with 23 loci having PIC values ​​greater than 0.5. HLJGY174 had the highest PIC value at 0.9275, while HLJGY143 had the lowest at 0.4897, classifying them as highly polymorphic markers. The above results collectively demonstrate that the 24 SSR marker combinations screened in this invention exhibit significant characteristics of high polymorphism, high heterozygosity, and high information content. They can provide accurate and reliable genetic parameters for germplasm identification of mandarin fish in the Heilongjiang River Basin, effectively meeting the practical application needs of germplasm identification.

[0129] Example 3: Germplasm identification of farmed mandarin fish populations

[0130] 1. Thirty samples of mandarin fish (original species likely originating from the Pearl River system, ZJ) from the Sanshui Shunhuayuan Aquatic Products Co., Ltd. in Foshan City, Guangdong Province, were analyzed. The average weight was (524.63±68.81) g and the average body length was (281.80±17.30) mm. Germplasm identification was performed based on the content of this invention.

[0131] 2. Phenotypic traits such as body length and height were measured. Statistical analysis revealed that the body length-to-height ratio of the Foshan Mandarin Fish population was 2.69–3.13, 2.84 ± 0.10; the body length-to-head length ratio was 2.56–3.20, with an average of 2.92 ± 0.18; the body length-to-tail peduncle length ratio was 6.60–10.35, with an average of 7.86 ± 0.79; the body length-to-tail peduncle height ratio was 8.01–9.71, with an average of 8.89 ± 0.40; and the head length-to-snout length ratio was 2.31–3.12, with an average of 2.68 ± 0.18. Significant differences were found in the body length-to-head length ratio, body length-to-tail peduncle height ratio, and head length-to-snout length ratio between the Foshan Mandarin Fish population (ZJ) and the Heilongjiang River Basin Mandarin Fish population (HLJ) (P < 0.05), demonstrating phenotypic differences between different geographical populations.

[0132] 3. Genetic parameters of the Foshan Mandarin Fish population were analyzed using six sets of germplasm discrimination primers. The population allele count ranged from 2 to 14, with an average of 6.417; observed heterozygosity ranged from 0.118 to 0.882, with an average of 0.656; expected heterozygosity ranged from 0.163 to 0.920, with an average of 0.667; and polymorphism information content ranged from 0.148 to 0.898, with an average of 0.615. Paired-samples t-tests were performed on the genetic parameters, and the results showed that the allele count, expected heterozygosity, and polymorphism information content of the Foshan population were significantly different from those of the Heilongjiang River basin population (P<0.05).

[0133] Table 4. Genetic discriminant parameters of Foshan mandarin fish (Pearl River Basin) population

[0134]

[0135] 4. The alleles and their frequencies at each marker locus were statistically analyzed. There were significant differences in the allele frequency distribution between the Pearl River Basin aquaculture population and the Heilongjiang River Basin population at all 24 SSR loci (Table 5). These differences were mainly reflected in three aspects: (1) Different allele types. The Pearl River aquaculture population detected only a few alleles at certain loci, while the Heilongjiang population had a richer variety of allele types. Conversely, some alleles that were missing in the Heilongjiang population were detected in the Pearl River population. For example, at the HLJGY202 locus, the Pearl River population mainly detected allele A, while the Heilongjiang population detected three alleles: A, B, and C. At the HLJGY122 locus, the Pearl River population detected three alleles: A, C, and D, while the Heilongjiang population detected six alleles: A, B, C, D, E, and F. This indicates that the allele pools of the two populations are significantly different. (2) Different dominant alleles. Among the alleles shared by the two groups, the most frequent dominant alleles are often inconsistent. For example, at the HLJGY143 locus, the dominant allele in the Pearl River group is C (frequency 0.7059), while the dominant allele in the Heilongjiang group is also C but with a frequency of only 0.4000. Moreover, the frequency of allele D (0.3833) in the Heilongjiang group is quite close to that of C, showing a dual dominant allele pattern. At the HLJGY033 locus, the dominant allele in the Pearl River group is A (0.3529), while the dominant alleles in the Heilongjiang group are J (0.1500) and A (0.1333), showing completely different dominant alleles. (3) The frequency difference of the same allele is significant. When comparing the alleles shared by the two groups, the frequency difference is generally large. Among them, the most significant differences were found at the following loci: allele A at HLJGY202, with a frequency of 0.9118 in the Pearl River population and only 0.3833 in the Heilongjiang population, a difference of 0.5285; allele C at HLJGY112, with a frequency of 0.4265 in the Pearl River population and 0.2000 in the Heilongjiang population, a difference of 0.2265; allele B at HLJGY241, with a frequency of 0.6765 in the Pearl River population and 0.2759 in the Heilongjiang population, a difference of 0.4006; and allele M at HLJGY095, with a frequency of 0.6176 in the Pearl River population and 0.3103 in the Heilongjiang population, a difference of 0.3073. The results showed that, based on the six sets of multiplex PCR primers described in this invention, the allele frequency profiles obtained from the detection of 24 SSR loci exhibited systematic and significant differences between the Pearl River aquaculture population and the Heilongjiang population. These differences were sufficient to clearly distinguish the two geographical populations. Therefore, if the allele frequency distribution characteristics of the tested sample at the aforementioned 24 loci are consistent with those of the Pearl River population in Table 1, but inconsistent with those of the Heilongjiang population, it can be determined that the tested sample did not originate from the Heilongjiang River basin.This embodiment fully verifies the effectiveness and practicality of the marker combination and multiplex PCR detection system of the present invention in the discrimination of cross-basin mandarin fish populations.

[0136] Table 5. Distribution of allele frequencies in aquaculture populations in Foshan and its comparison with those in the Heilongjiang River Basin.

[0137]

[0138]

Claims

1. A method for identifying germplasm of Mandarin fish from the Heilongjiang River basin, characterized in that, The method for identifying the germplasm of Mandarin fish in the Heilongjiang River Basin is carried out according to the following steps: I. Collect parental samples of Mandarin fish from the Heilongjiang River basin; II. Perform phenotypic data analysis on the collected samples; III. Extract DNA from the collected samples and analyze the genotypes of the parent individuals using a combination of six SSR marker primers to establish fingerprint patterns and genetic parameter ranges. The names of the SSR markers, primer sequences, and amplification combinations are as follows: Multiplex PCR group I: HLJ GY222 Upstream primer: ROX-5'-GATCA CCTAA ACCCA GACAC C-3', as shown in SEQ ID NO:

1. Downstream primer: 5'-ATTTC CACCA TGATG CCAGC-3', as shown in SEQ ID NO:

2. HLJGY134 Upstream primer: FAM-5'-GATAA GATGC CGTCA TGTGC-3', as shown in SEQ ID NO:

3. Downstream primer: 5'-TCAAA CATAG GGCTC AACTG G-3', as shown in SEQ ID NO:

4. HLJGY213 Upstream primer: HEX-5'-ACTTA CTGAC AGAAG CGTGC-3', as shown in SEQ ID NO:

5. Downstream primer: 5'-CTGAT AGTGA CGTCA TCGCC-3', as shown in SEQ ID NO:

6. HLJGY064 Upstream primer: TAMRA-5'-AGGTG ACGCA CAATT AGACC-3', as shown in SEQ ID NO:

7. Downstream primer: 5'-GATAC ACTGC AGCCC ATAGG-3', as shown in SEQ ID NO:

8. Multiplex PCR group II: HLJGY122 Upstream primer: ROX-5'-ATCAC CCAAC TGCAG ATTCC-3', as shown in SEQ ID NO:

9. Downstream primer: 5'-TCCAC AGCTA AGTTA GTCGC-3', as shown in SEQ ID NO:

10. HLJGY112 Upstream primer: FAM-5'-TCTTT ACCTG GAGAG GACGG-3', as shown in SEQ ID NO:

11. Downstream primer: 5'-GGCAT GTTAT GGTCA GGAGG-3', as shown in SEQ ID NO:

12. HLJGY202 Upstream primer: HEX-5'-AAGTC TCGTT GTGGT GATGG-3', as shown in SEQ ID NO:

13. Downstream primer: 5'-TCAGG AGGAG CAAAC AAAGG-3', as shown in SEQ ID NO:

14. HLJGY033 Upstream primer: TAMRA-5'-TGTGA TGAGA TGCCT TGTCG-3', as shown in SEQ ID NO:

15. Downstream primer: 5'-ATATA CACCA CTGCC ACAGG-3', as shown in SEQ ID NO:

16. Multiplex PCR group III: HLJGY043 Upstream primer: ROX-5'-TTTCT GGGCT GGGAT TTAGG-3', as shown in SEQ ID NO:

17. Downstream primer: 5'-TATGC TCTAC CATGC ACTCG-3', as shown in SEQ ID NO:

18. HLJGY182 Upstream primer: FAM-5'-GGCTC GCTAT TGAAC ATGG-3', as shown in SEQ ID NO:

19. Downstream primer: 5'-GTTGA AGAGG TGGTT CCAGG-3', as shown in SEQ ID NO:

20. HLJGY084 Upstream primer: HEX-5'-CAACG TAGGT ATTGC ACTGC-3', as shown in SEQ ID NO:

21. Downstream primer: 5'-AATCG GCATG TCTCT TCTGC-3', as shown in SEQ ID NO:

22. HLJGY163 Upstream primer: TAMRA-5'-TGGTC ACTTA CACAC AGTGG-3', as shown in SEQ ID NO:

23. Downstream primer: 5'-TCTGA AATGG GCCAT TCTGC-3', as shown in SEQ ID NO:

24. Multiplex PCR group IV: HLJ GY241 Upstream primer: ROX-5'-CAAGT AGTGC CACCA AGAGG-3', as shown in SEQ ID NO:

25. Downstream primer: 5'-TCGAC TCTGA ACAAT AGCTG C-3', as shown in SEQ ID NO:

26. HLJGY016 Upstream primer: FAM-5'-TATGA AGGCC TATGT GCACG-3', as shown in SEQ ID NO:

27. Downstream primer: 5'-GCGAT GTTGT GTTAC TCTCG-3', as shown in SEQ ID NO:

28. HLJGY174 Upstream primer: HEX-5'-ATGCT TTCTG TGTGA CCTCC-3', as shown in SEQ ID NO:

29. Downstream primer: 5'-CGGAT GCACA CTTAT AAACA GC-3', as shown in SEQ ID NO:

30. HLJGY105 Upstream primer: TAMRA-5'-TTCTT GCTCT CACAC ACTCC-3', as shown in SEQ ID NO:

31. Downstream primer: 5'-AGATA GATAG ATGGA CGGAC G-3', as shown in SEQ ID NO:

32. Multiplex PCR group V: HLJGY151 Upstream primer: ROX-5'-GACCT ACAGT AGACC TCAAG C-3', as shown in SEQ ID NO:

33. Downstream primer: 5'-GCTGT AGCAC TGCAA TTTCC-3', as shown in SEQ ID NO:

34. HLJGY074 Upstream primer: FAM-5'-TGGAT GAACC TTGCT TTAGG G-3', as shown in SEQ ID NO:

35. Downstream primer: 5'-TCCCT TTGCA GAAGA ATCGC-3', as shown in SEQ ID NO:

36. HLJGY054 Upstream primer: HEX-5'-AGTAG AGACA ACAGC AACGG-3', as shown in SEQ ID NO:

37. Downstream primer: 5'-TCATG CTGTA ACTCT GCTCG-3', as shown in SEQ ID NO:

38. HLJGY021 Upstream primer: TAMRA-5'-AAGCT GGAGA GATGT TTGGG-3', as shown in SEQ ID NO:

39. Downstream primer: 5'-AGGAG GAAGG GTACT ACTTG G-3', as shown in SEQ ID NO:

40. Multiplex PCR VI: HLJ GY231 Upstream primer: ROX-5'-TGGAC AGCAG TGTAT GTTGG-3', as shown in SEQ ID NO:

41. Downstream primer: 5'-CAAGG GTGTT CAAGG GTAGG-3', as shown in SEQ ID NO:

42. HLJGY143 Upstream primer: FAM-5'-TCATC TGCAG GTCTG TTTGG-3', as shown in SEQ ID NO:

43. Downstream primer: 5'-TGCAG ACTGG CACAA TTCC-3', as shown in SEQ ID NO:

44. HLJGY095 Upstream primer: HEX-5'-GACTT CACAG AAGCA AAGCC-3', as shown in SEQ ID NO:

45. Downstream primer: 5'-CCAAG CTTTG TTCAC ACAGC-3', as shown in SEQ ID NO:

46. HLJGY192 Upstream primer: TAMRA-5'-GATAT GAGGC AACAC ACAGC-3', as shown in SEQ ID NO:

47. Downstream primer: 5'-ACCCT CACAC TTTGT CTTAA CC-3'; as shown in SEQ ID NO:

48. IV. Identify the germplasm of Heilongjiang mandarin fish by combining phenotypic parameters, fingerprint pattern maps, and genetic parameters.

2. The method for identifying the germplasm of Mandarin fish in the Heilongjiang River Basin according to claim 1, characterized in that, The phenotypic data in step two include the individual measurements of the mandarin fish: weight, total length, body length, body height, head length, snout length, caudal peduncle height, and caudal peduncle length.

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