Portunus trituberculatus paternity test and population genetic analysis method

Abstract

According to the portunus trituberculatus paternity test and population genetic analysis method provided by the invention, portunus trituberculatus primers are screened for test combination to form multiple groups of multiple PCR amplification systems, and primer combinations with different lengths are selected, so that the effects that multiple primers do not influence one another, non-specific amplification is not generated, and amplification products are not overlapped can be realized. In the PCR amplification process, a plurality of target fragments are amplified at the same time, so that the purposes of saving experimental samples and experimental cost and improving experimental efficiency can be achieved, and the paternity test can be accurately performed on the portunus trituberculatus. The fluorescent microsatellite multiplex PCR paternity test method for the portunus trituberculatus can be applied to the fields of genetic relationship identification, population genetic analysis, genetic linkage map construction and molecular assisted breeding of the portunus trituberculatus.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology DNA markers, and particularly relates to a method for parentage identification and population genetic analysis of swimming crabs (Portunus trituberculatus). Background Art

[0002] The three-spotted swimming crab (Portunus trituberculatus), also known as the pistol crab or three-spotted crab, belongs to the phylum Arthropoda, class Crustacea, order Decapoda, order Brachyura, family Portunidae, and genus Portunus. The three-spotted swimming crab has a wide distribution range, found in the waters of China, North Korea, South Korea, Japan, and the Malaysian archipelago. In my country, it is primarily found in the Yellow Sea, Bohai Sea, East China Sea, and South China Sea. Its aquaculture and fishing yields a significant amount, accounting for 90% of the national total.

[0003] The swimming crab (Swinhoe crab) boasts advantages such as rapid growth, large size, delicious flavor, and rapid sexual maturity. Consequently, it enjoys high market demand and is a major aquaculture and fishing species in my country. Therefore, the selection of selected varieties with specific desirable traits is crucial for furthering the development of the swimming crab aquaculture industry. However, inbreeding between selected varieties and conventionally farmed swimming crabs is inevitable during artificial cultivation. Over generations of directional selection, this can lead to undesirable phenomena such as the deterioration of desirable traits, reduced survival rates, and decreased genetic diversity. This can lead to a decline in the swimming crab germplasm. Therefore, genetic analysis of populations during the cultivation of swimming crab is crucial.

[0004] DNA molecular markers are polymorphic fragments that can reveal variations in nucleotide sequences within an organism. These fragments are numerous and possess stable inheritance characteristics, unaffected by environmental or gene expression changes. With the continuous advancement of biotechnology, DNA molecular markers have become one of the more accurate and reliable genetic marker technologies. Molecular marker technology has been widely used in genetic analysis of aquatic animals, providing fundamental data for monitoring population stability. Research on molecular marker-assisted breeding is of great significance in improving and enhancing the artificial cultivation and genetic breeding of swimming crabs (Portunus trituberculatus).

[0005] Microsatellite markers are evenly distributed throughout the genome, are numerous, have few repetitive sequences, and exhibit good reproducibility, making them an excellent tool for studying gene function. They are highly polymorphic and well conserved; possess numerous alleles, are highly specific at their loci, and offer a wealth of information. They can simultaneously analyze genetic variation in multiple traits. According to Mendel's laws of inheritance, they exhibit codominant inheritance, clearly distinguish between heterozygotes and homozygotes, and detect single alleles. Because SSR molecular marker technology amplifies shorter fragments, template DNA extraction requirements are less stringent. Furthermore, the technology's workflow is simple, and the detection process is convenient and rapid. Furthermore, its results are stable and reproducible. Consequently, microsatellite markers have become the most commonly used molecular marker technology.

[0006] Multiplex polymerase chain reaction (PCR) involves adding two or more pairs of primers to a reaction, allowing for the amplification of multiple sequences in a single reaction. This technique has been combined with microsatellite markers to form fluorescent microsatellite multiplex PCR, in which fluorescent tags are added to the 5' ends of different primers within a single system. This facilitates primer reading and enhances genotyping. This method is highly efficient, avoids sample waste, and significantly reduces experimental time and costs. Therefore, it has been widely used in paternity testing and genetic diversity analysis. Fluorescent microsatellite multiplex PCR for swimming crabs (P. trituberculatus) could provide technical support for future efforts in breeding superior lineages, investigating germplasm, and conserving the species. Summary of the Invention

[0007] The present invention aims to provide a method for paternity testing and population genetic analysis of the swimming crab (Portunus trituberculatus). By screening primers for the swimming crab (Portunus trituberculatus) and experimentally combining multiple multiplex PCR amplification systems, and selecting primer combinations of different lengths, the present invention can achieve the effects of eliminating mutual influence between the multiple primers, preventing nonspecific amplification, and preventing overlap of amplified products. During the PCR amplification process, multiple target fragments are amplified simultaneously, thereby saving experimental samples, experimental costs, and improving experimental efficiency. Furthermore, the present invention can accurately perform paternity testing on the swimming crab (Portunus trituberculatus).

[0008] The present invention first provides a primer pair combination for multiplex PCR amplification of microsatellites of Portunus trituberculatus, wherein one primer pair combination comprises the following primer pairs:

[0009] The SSR28-3 primer pair has an upstream primer sequence of 5′-TTTCTTTCACGGCTACGGGC-3′ (SEQ ID NO: 1) and a downstream primer sequence of 5′-TGCATGTATGACTTTAGGCGC-3′ (SEQ ID NO: 2);

[0010] The SSR25-1 primer pair has an upstream primer sequence of 5′-CCCCCTTTGTCTTATCCCCG-3′ (SEQ ID NO: 3) and a downstream primer sequence of 5′-CTGCAGGGGAACAATGGAGG-3′ (SEQ ID NO: 4);

[0011] The SSR114 primer pair has an upstream primer sequence of 5′-CTGTCTGTGACACCAATGCG-3′ (SEQ ID NO: 5) and a downstream primer sequence of 5′-CATTTACCCATGCAGGTGCG-3′ (SEQ ID NO: 6);

[0012] The SSR118 primer pair has an upstream primer sequence of 5′-TCATGGTAAGGCAGAAGCGG-3′ (SEQ ID NO: 7) and a downstream primer sequence of 5′-TGTGCTGATGTCACTCGTGC-3′ (SEQ ID NO: 8);

[0013] Another primer pair combination for microsatellite multiplex PCR amplification provided by the present invention includes the following primer pairs:

[0014] The SSR38-3 primer pair has an upstream primer sequence of 5′-TCTTATTTACCTCTCTCTCTCTCTCG-3′ (SEQ ID NO: 9) and a downstream primer sequence of 5′-GAAGGGTGTCCACAGTCTGC-3′ (SEQ ID NO: 10);

[0015] The SSR40-2 primer pair has an upstream primer sequence of 5′-GATCCCCGCAATCTACCACC-3′ (SEQ ID NO: 11) and a downstream primer sequence of 5′-CATTGTATGACGGCAAGGGC-3′.

[0016] (SEQ ID NO: 12);

[0017] The SSR21-2 primer pair has an upstream primer sequence of 5′-TCTTCGTGACAGAGGCTTGC-3′ (SEQ ID NO: 13) and a downstream primer sequence of 5′-CACGGCACAATACTGAACGC-3′ (SEQ ID NO: 14);

[0018] The SSR112 primer pair has an upstream primer sequence of 5′-ACATCCATCATTTCCCCAGCC-3′ (SEQ ID NO: 15) and a downstream primer sequence of 5′-TCACCACCATCATCACTGCC-3′.

[0019] (SEQ ID NO: 16);

[0020] The KX-61 primer pair has an upstream primer sequence of 5′-GACGCAGACGAAGGCGGATCTTATTAGC-3′ (SEQ ID NO: 17) and a downstream primer sequence of 5′-GGGATGGAAGTCATTGTTAGTATCGTCAAAGGAAG-3′ (SEQ ID NO: 18);

[0021] Another primer pair combination for microsatellite multiplex PCR amplification provided by the present invention includes the following primer pairs:

[0022] The SSR 6-3 primer pair has an upstream primer sequence of 5′-CTGTGCACACGTTGTCATGC-3′ (SEQ ID NO: 19) and a downstream primer sequence of 5′-CACATGGATCTGGTTCCCCG-3′.

[0023] (SEQ ID NO: 20);

[0024] The SSR19-3 primer pair has an upstream primer sequence of 5′-AAGTGGCTTCCTGATGGAGC-3′ (SEQ ID NO: 21) and a downstream primer sequence of 5′-CTCACGTGTGCTTGCTTGC-3′ (SEQ ID NO: 22);

[0025] The SSR45-1 primer pair has an upstream primer sequence of 5′-TCTCTGAAGTGGAAGCGACG-3′ (SEQ ID NO: 23) and a downstream primer sequence of 5′-ATATCTCCACCAGGCCGAGG-3′ (SEQ ID NO: 24);

[0026] The SSR1-3 primer pair has an upstream primer sequence of 5′-GGTTGTACGTGGCAAAGAGC-3′ (SEQ ID NO: 25) and a downstream primer sequence of 5′-GTCTCTCCTTGTGTTGTTTGCC-3′ (SEQ ID NO: 26);

[0027] The SSR117 primer pair has an upstream primer sequence of 5′-CCCTACAGAGATGTGAGGCG-3′ (SEQ ID NO: 27) and a downstream primer sequence of 5′-TTTTGTCTCGAGTCAGGGCG-3′.

[0028] (SEQ ID NO: 28);

[0029] The SSR34-1 primer pair has an upstream primer sequence of 5′-TTTTTCTTCGGTCGTTTTGGC-3′ (SEQ ID NO: 29) and a downstream primer sequence of 5′-TATCAAGCACCAAGTTTCCGC-3′ (SEQ ID NO: 30);

[0030] The SSR33-1 primer pair has an upstream primer sequence of 5′-GGGTACAATGAAGCTCAGCG-3′ (SEQ ID NO: 31) and a downstream primer sequence of 5′-ACAATAAGAAAGTGAACACGCGG-3′ (SEQ ID NO: 32);

[0031] The SSR42-3 primer pair has an upstream primer sequence of 5′-AGAAGAGAAAGTAATGAAAGAATCACG-3′ (SEQ ID NO: 33) and a downstream primer sequence of 5′-CCTGTGTGACAATTAGCGGC-3′ (SEQ ID NO: 34);

[0032] The SSR19-2 primer pair has an upstream primer sequence of 5′-AGCATGTACACCAGAAGGACG-3′ (SEQ ID NO: 35) and a downstream primer sequence of 5′-CTTACGTGGGAGACAGAGAGC-3′ (SEQ ID NO: 36);

[0033] The SSR119 primer pair has an upstream primer sequence of 5′-TCCCTCACAAACACACCTGC-3′ (SEQ ID NO: 37) and a downstream primer sequence of 5′-TCTCTGTATAAAAAGGTGCACGC-3′ (SEQ ID NO: 38).

[0034] Furthermore, the primer pair carries a fluorescent label at its 5' end or 3' end.

[0035] The present invention also provides a use of the microsatellite multiplex PCR amplification primer pair combination, which is used in detecting the genetic diversity of swimming crab (Portunus trituberculatus);

[0036] In another aspect, the present invention provides another use of the microsatellite multiplex PCR amplification primer pair combination, which is the use in parentage testing of swimming crabs (Portunus trituberculatus).

[0037] The present invention also provides a method for parentage identification of swimming crabs (Portunus trituberculatus), wherein the method uses the above-mentioned microsatellite multiplex PCR amplification primer pair for identification.

[0038] The present invention provides a paternity testing method for the swimming crab (Portunus trituberculatus) using fluorescent microsatellite multiplex PCR. By performing paternity testing analysis on parent samples, the method achieves a 100% paternity testing success rate for all 10 parents and 150 offspring at a 95% confidence level. The method can be used for kinship identification, population genetic analysis, construction of genetic linkage maps, and molecular-assisted breeding of the swimming crab. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :Results of DNA extraction from the swimming crab genome;

[0040] Figure 2 : Denaturing polyacrylamide gel electrophoresis results of primers SSR35-4 and SS36-3;

[0041] Figure 3 : Genotyping peaks of four groups of microsatellites of Portunus trituberculatus by multiplex PCR system; wherein, Figure A is combination PTM1-4; Figure B is combination PTM2-5; Figure C is combination PTM3-5; Figure D is combination PTM4-5;

[0042] Figure 4 :Cluster diagram of individual families of swimming crab (Portunus trituberculatus);

[0043] Figure 5 : UPGMA cluster diagram of Portunus trituberculatus population based on Nei's genetic distance. DETAILED DESCRIPTION

[0044] The swimming crabs used in this example were sourced from 25 brooding female Swimming Crabs (M. trituberculatus) and their hatched offspring, stage I zoeae, bred at the Lanshang Marine Technology Company farm in Xiangshan County. Samples were obtained from the ventral limb muscles of the mothers, and from the offspring, stage I zoeae. Both samples were preserved in alcohol and frozen at -20°C for DNA extraction and labeling. Ultimately, 10 sets of parent-offspring samples—one mother and her 15 offspring—were selected for subsequent experimental validation.

[0045] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0046] Example 1: Screening of primer combinations for microsatellite fluorescence multiplex PCR amplification of parentage identification of Portunus trituberculatus

[0047] The method for paternity testing of swimming crab (Portunus trituberculatus) by microsatellite fluorescence multiplex PCR provided in this embodiment comprises the following steps:

[0048] 1) DNA extraction from Portunus trituberculatus

[0049] Ten groups of parent and offspring samples of Swimming crab (Portunus trituberculatus), including the muscle of the mother and its 15 first-stage zoeae, were immediately stored in anhydrous ethanol. Genomic DNA was extracted using the OMEGA Tissue DNA Kit, and the DNA concentration was adjusted to 30 ng / μl. The results of genomic DNA extraction from Swimming crab (Portunus trituberculatus) are shown in Figure 2. Figure 1 shown.

[0050] 2) Screening of microsatellite primers for Portunus trituberculatus

[0051] Based on the genome data of the swimming crab (Portunus trituberculatus) downloaded from NCBI, we used Krait software to screen microsatellite loci and design primers, ensuring that there was a large difference in the length of the amplified fragments. The primers were sent to our company for synthesis. The polymorphism and stability of the microsatellite loci were detected by 1.5% agarose gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and capillary electrophoresis. Ultimately, primers with high polymorphism, stable amplification, and strong specificity were selected. The 5′ end of the primers was fluorescently modified. The selected primer pairs are as follows:

[0052] The SSR28-3 primer pair has an upstream primer sequence of 5′-TTTCTTTCACGGCTACGGGC-3′ (SEQ ID NO: 1) and a downstream primer sequence of 5′-TGCATGTATGACTTTAGGCGC-3′ (SEQ ID NO: 2);

[0053] The SSR25-1 primer pair has an upstream primer sequence of 5′-CCCCCTTTGTCTTATCCCCG-3′ (SEQ ID NO: 3) and a downstream primer sequence of 5′-CTGCAGGGGAACAATGGAGG-3′ (SEQ ID NO: 4);

[0054] The SSR114 primer pair has an upstream primer sequence of 5′-CTGTCTGTGACACCAATGCG-3′ (SEQ ID NO: 5) and a downstream primer sequence of 5′-CATTTACCCATGCAGGTGCG-3′ (SEQ ID NO: 6);

[0055] The SSR118 primer pair has an upstream primer sequence of 5′-TCATGGTAAGGCAGAAGCGG-3′ (SEQ ID NO: 7) and a downstream primer sequence of 5′-TGTGCTGATGTCACTCGTGC-3′ (SEQ ID NO: 8);

[0056] The SSR38-3 primer pair has an upstream primer sequence of 5′-TCTTATTTACCTCTCTCTCTCTCTCG-3′ (SEQ ID NO: 9) and a downstream primer sequence of 5′-GAAGGGTGTCCACAGTCTGC-3′ (SEQ ID NO: 10);

[0057] The SSR40-2 primer pair has an upstream primer sequence of 5′-GATCCCCGCAATCTACCACC-3′ (SEQ ID NO: 11) and a downstream primer sequence of 5′-CATTGTATGACGGCAAGGGC-3′.

[0058] (SEQ ID NO: 12);

[0059] The SSR21-2 primer pair has an upstream primer sequence of 5′-TCTTCGTGACAGAGGCTTGC-3′ (SEQ ID NO: 13) and a downstream primer sequence of 5′-CACGGCACAATACTGAACGC-3′ (SEQ ID NO: 14);

[0060] The SSR112 primer pair has an upstream primer sequence of 5′-ACATCCATCATTTCCCCAGCC-3′ (SEQ ID NO: 15) and a downstream primer sequence of 5′-TCACCACCATCATCACTGCC-3′.

[0061] (SEQ ID NO: 16);

[0062] The KX-61 primer pair has an upstream primer sequence of 5′-GACGCAGACGAAGGCGGATCTTATTAGC-3′ (SEQ ID NO: 17) and a downstream primer sequence of 5′-GGGATGGAAGTCATTGTTAGTATCGTCAAAGGAAG-3′ (SEQ ID NO: 18);

[0063] The SSR 6-3 primer pair has an upstream primer sequence of 5′-CTGTGCACACGTTGTCATGC-3′ (SEQ ID NO: 19) and a downstream primer sequence of 5′-CACATGGATCTGGTTCCCCG-3′.

[0064] (SEQ ID NO: 20);

[0065] The SSR19-3 primer pair has an upstream primer sequence of 5′-AAGTGGCTTCCTGATGGAGC-3′ (SEQ ID NO: 21) and a downstream primer sequence of 5′-CTCACGTGTGCTTGCTTGC-3′ (SEQ ID NO: 22);

[0066] The SSR45-1 primer pair has an upstream primer sequence of 5′-TCTCTGAAGTGGAAGCGACG-3′ (SEQ ID NO: 23) and a downstream primer sequence of 5′-ATATCTCCACCAGGCCGAGG-3′ (SEQ ID NO: 24);

[0067] The SSR1-3 primer pair has an upstream primer sequence of 5′-GGTTGTACGTGGCAAAGAGC-3′ (SEQ ID NO: 25) and a downstream primer sequence of 5′-GTCTCTCCTTGTGTTGTTTGCC-3′ (SEQ ID NO: 26);

[0068] The SSR117 primer pair has an upstream primer sequence of 5′-CCCTACAGAGATGTGAGGCG-3′ (SEQ ID NO: 27) and a downstream primer sequence of 5′-TTTTGTCTCGAGTCAGGGCG-3′.

[0069] (SEQ ID NO: 28);

[0070] A preliminary screening of 200 primer pairs was conducted, and the primers with clear bands and strong specificity in the preliminary screening were screened by polyacrylamide gel electrophoresis. The microsatellite primers selected in the preliminary screening were screened again using 18 wild swimming crab DNA samples. Finally, 43 pairs of highly polymorphic sites were identified, and the amplification effects of some of the sites were as follows: Figure 2 shown.

[0071] Fluorescently labeled combinations. Forty-three primers were labeled with blue fluorescent markers and screened by capillary electrophoresis. The results showed that 32 primers exhibited good fluorescence patterns after adding fluorescent markers, with high peaks, typically with a single peak or a distinct main peak, indicating good polymorphism. These 32 primer pairs were subsequently used for combined screening in multiplex PCR systems.

[0072] 3) Construction of multiplex PCR amplification system

[0073] The primers screened in step 2) were preliminarily grouped according to the size of the PCR product fragments after amplification. Based on the principle that the amplified bands were clear and the amplified product size ranges did not overlap, they were first combined in pairs. Primers that met the requirements were further added. Ultimately, 19 microsatellite loci were combined into four multiplex PCR systems, namely one quadruple PCR amplification primer combination and three quintuple PCR amplification primer combinations.

[0074] The names of the combinations and the primer pairs they contain are as follows:

[0075] 1)PTM1-4: SSR28-3, SSR25-1, SSR114, SSR118;

[0076] 2)PTM2-5: SSR38-3, SSR40-2, SSR21-2, SSR112, KX-61;

[0077] 3)PTM3-5: SSR 6-3, SSR19-3, SSR45-1, SSR1-3, SSR117;

[0078] 4)PTM4-5: SSR34-1, SSR33-1, SSR42-3, SSR19-2, SSR119;

[0079] The sequences and detailed parameters of each multiplex PCR primer pair are shown in Table 2. In the random samples of swimming crab (Portunus trituberculatus), the amplification effects of the four multiplex PCR systems are as follows: Figure 3 exhibit.

[0080] Table 1: Multiplex PCR information of five groups of microsatellites in Portunus trituberculatus

[0081]

[0082]

[0083] 4) Paternity testing

[0084] The multiplex PCR system constructed in step 3) amplified the genomic DNA from the parental and offspring samples described in step 1). The amplification conditions for the sample genomic DNA were: 94°C pre-denaturation for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, and extension at 72°C for 10 minutes. Multiplex PCR products were then sequenced using an ABI 3730XL capillary electrophoresis instrument for STR typing. Allele analysis was performed using Genemapper 4.0 software to obtain genotype data for each locus in the parents and offspring, and parent-offspring relationships were determined according to Mendel's laws. The genetic diversity of the products amplified by each primer combination is shown in Table 2.

[0085] Table 2: Biodiversity information of microsatellite multiplex PCR amplification primer pairs

[0086]

[0087]

[0088] Note: Sample size: 160; Na: number of alleles; Ho: observed heterozygosity; He: expected heterozygosity; HW: Hardy-Weinberg equilibrium. NS: not significant; ND: not certain; *: significance level 5%; **: significance level 1%; ***: significance level 0.1%.

[0089] The results showed that the 19 pairs of microsatellite primers used for screening had high polymorphism and could be fully and effectively used in the parentage identification system of swimming crab (Portunus trituberculatus).

[0090] The effects of paternity testing on the 19 pairs of microsatellite primer pairs screened in this example were analyzed.

[0091] 1) Parental exclusion probability analysis

[0092] Using a specific genetic marker to determine the probability that a parent is the biological parent of the offspring to be identified can be used to evaluate the effectiveness of microsatellite markers in paternity testing. Because microsatellite markers can distinguish between paternal and maternal parents, they can be used to effectively identify offspring. In this experiment, when information about both parents is unclear, the relationship between offspring and candidate maternal parents was determined, and the exclusion probability was calculated when parental information is unclear. First, the exclusion probability for each microsatellite locus was calculated based on the allele frequencies of 19 microsatellite loci in four multiplex PCR systems. Then, the cumulative exclusion probability for all 19 loci was calculated. The results are shown in Table 3. As can be seen from Table 3, when the genotypes of both parents are unclear, the parental exclusion probability for each of the 19 microsatellite loci ranged from 7.5% to 72.5%, and the cumulative exclusion probability reached 100%. The cumulative exclusion rate for all 19 microsatellite loci exceeded 100% in the four multiplex PCR systems, providing a powerful tool for paternity testing in the swimming crab (Portunus trituberculatus).

[0093] Table 3: Data table of exclusion probability and cumulative exclusion probability of paternity test of swimming crab (Portunus trituberculatus)

[0094]

[0095]

[0096] 2) Analysis of parent-child relationship distribution

[0097] Cervus software was used to simulate four multiplex PCR combinations, with 10,000 progeny and 20 parents each. The identification rates of the combinations were calculated under four models: known maternal sex, known paternal sex, unknown parental sex, and known parental sex. Simulation analysis of each combination revealed that the identification efficiency of combinations PTM4-5 was high in all four models, at 100%, 100%, 85%, and 100%, respectively. The identification efficiency of combinations PTM1-4 was relatively low, at 53%, 62%, and 92%, respectively. Simulations of any two or more combinations achieved a 100% identification rate across all four models.

[0098] The Colony software was used to perform actual paternity testing on 10 maternal parents and 150 offspring, with a confidence interval of 95% and an error of 0.1%. The analysis results showed that all 150 offspring could be matched to their corresponding maternal parents, and the matching assignment rate and accuracy rate were both 100%. Figure 4 As shown in the figure, 10 maternal parents and 150 offspring were subjected to UPGMA cluster analysis, and the results showed that all families could be clustered into one category.

[0099] Example 2: Analysis of genetic diversity in different populations of swimming crab (Portunus trituberculatus)

[0100] 1. Multiplex PCR polymorphism analysis

[0101] Genetic diversity analysis among eight different populations of swimming crab (P. trituberculatus) was conducted using two combinations of stably amplified and highly polymorphic SSR loci (PTM2-5 and PTM3-5). The results, shown in Table 5, show that a total of 307 alleles (Na) were detected using Cervus, with an average of 31 alleles per locus. KX-61 had the highest number of alleles (Na), with 49, followed by SSR1-3, with 44 alleles. SSR19-3 had the lowest number of alleles (Na), with 11. The effective number of alleles (Ne) for the 10 SSR loci ranged from 1.774 to 25.982, with an average of 13.977. KX-61 had the highest effective number of alleles (Ne), with 26, while SSR38-3 had the lowest number of alleles (Ne), with only 1.774. The observed heterozygosity (Ho) ranged from 0.335 to 0.935, with an average of 0.746. The highest observed heterozygosity (Ho) of KX-61 was 0.935, and the lowest observed heterozygosity (Ho) of SSR38-3 was 0.335. The expected heterozygosity (He) ranged from 0.437 to 0.964, with an average of 0.874. Similar to the observed heterozygosity (Ho), the highest and lowest expected heterozygosities (He) were also KX-61 and SSR38-3, with expected heterozygosities (He) of 0.964 and 0.437, respectively. The polymorphic information content (PIC) ranged from 0.411 to 0.96, with an average of 0.864. The highest PIC was 0.96 at the KX-61 locus, and the lowest was 0.411 at SSR38-3. The average PIC of each locus was greater than 0.5, indicating that the two multiplex PCR combinations were highly polymorphic.

[0102] Table 4: Sample information table

[0103]

[0104] Table 5: Genetic diversity parameters of microsatellite loci

[0105]

[0106]

[0107] Note: Na: number of alleles; Ne: effective number of alleles; Ho: observed heterozygosity; He: expected heterozygosity; PIC: polymorphic information content; HW: Havenberg equilibrium; ND: not done; NS: not significant; *: significance level 5%; **: significance level 1%; ***: significance level 0.1%;

[0108] 2. Genetic diversity analysis of eight populations of swimming crab (Portunus trituberculatus)

[0109] The results of genetic diversity analysis of the eight populations are shown in Table 6. The average number of alleles (Na) of the eight populations ranged from 9.100 to 16.600, among which the wild population had the highest average number of alleles (Na) of 16.600, and the XG-2 farmed population had the lowest Na value of 9.100.

[0110] The average effective allele number (Ne) of each population ranged from 4.623 to 10.755, and the population with the highest effective allele number (Ne) was the wild population, with the highest value of 10.755, and the population with the lowest effective allele number (Ne) was XG-2, with the lowest value of 4.623.

[0111] The average Xiangnong diversity index (I) ranged from 1.722 to 2.432. The wild population had the highest Xiangnong diversity index (I), reaching 2.432. The Xiangnong diversity index (I) of the two populations in the XG culture pond was lower, with the XG-1 culture population having the lowest Xiangnong diversity index (I) of 1.722, followed by the XG-2 culture population at 1.915.

[0112] The mean observed heterozygosity (Ho) ranged from 0.691 to 0.810, while the mean expected heterozygosity (He) ranged from 0.759 to 0.863. FM-1 and XG-2 had lower mean observed heterozygosity (Ho), at 0.691 and 0.698, respectively, while FS-2 had a higher Ho, at 0.81. The expected heterozygosity (He) of FM-1, FS-1, and the wild-type populations was 0.863, 0.862, and 0.860, respectively, with XG-1 having the lowest expected heterozygosity (He). With the exception of FS-2, all other populations showed expected heterozygosity (He) greater than observed heterozygosity (Ho).

[0113] The average polymorphic information content (PIC) ranged from 0.729 to 0.85. The XG-1 population had the lowest PIC, at 0.729. The wild populations, FM-1, and FS-1 all had higher PICs, with an average of 0.85. The inbreeding coefficient (Fis) for all loci across the two populations ranged from -0.046 to 0.322, with an average of 0.084. The overall inbreeding coefficient (Fit) ranged from -0.041 to 0.408, with an average of 0.137.

[0114] Comparison of genetic diversity among different populations revealed that, with the exception of Ho, all genetic diversity parameters of wild swimming crab (S. trituberculatus) were higher than those of the Ningxiang No. 1 population and the three farmed populations. The genetic diversity parameters of the XG pond population were all lower. The values ​​of Na, Ne, and I showed a consistent trend across the eight populations, with the order Y > N1 > FS-2 > FM-2 > XG-2. The values ​​of He and PIC showed a consistent trend across the five adult populations, with the order Y > N1 > FS-2 > XG-2 > FM-2. The polymorphic information content (PIC) of all eight populations was high, exceeding 0.7, with an average of 0.8. Comparison of genetic diversity among three juvenile swimming crab populations (FS-1, FM-1, and XG-1) and three farmed adult swimming crab populations (FS-2, FM-2, and XG-2) revealed that, with the exception of the XG pond population, the juvenile populations exhibited higher genetic diversity than the adult populations in all other two pond populations.

[0115] Table 6: Genetic diversity parameters of eight different populations of swimming crab (Portunus trituberculatus)

[0116]

[0117]

[0118] 3. Genetic differentiation and genetic distance among five populations of adult swimming crabs

[0119] Genetic differentiation indices (Fst) were calculated for five populations of adult swimming crabs (Y, N, FS-2, FM-2, and XG-2). The results are shown in Table 7. The genetic differentiation indices (Fst) between populations ranged from 0.02 to 0.051. The genetic differentiation indices (Fst) between XG-2, N1, and FM-2 were no less than 0.05, at 0.051 and 0.05, respectively. The genetic differentiation indices (Fst) between the remaining populations were all less than 0.05. Among them, the genetic differentiation indices for FM-2 and FS-2 were relatively low, at 0.02. Gene flow (Nm) among the five populations of adult swimming crabs ranged from 3.99 to 12.76, indicating a high level of gene exchange between the populations.

[0120] As shown in Table 8, the AMOVA analysis of the five populations of swimming crabs found that genetic variation mainly existed between individuals (79%), followed by between individuals within a population (15%), while genetic differentiation between populations was less, accounting for only 6%.

[0121] As shown in Table 9, the genetic distances of the five adult swimming crab populations ranged from 0.167 to 0.572. The genetic distance between the XG-2 and N1 populations was the longest at 0.72, and the genetic similarity was the lowest at 0.564. The genetic distance between the FS-2 and FM-2 populations was the smallest at 0.167, and the genetic similarity was the largest at 0.846. Figure 5It can be seen that the ordinary breeding groups are clustered into one branch, and the wild groups and the breeding groups are clustered into one branch.

[0122] Table 7: Genetic differentiation index (Fst) and significance (P) of five populations of adult swimming crabs

[0123]

[0124] Table 8: Analysis of variance of five populations of adult swimming crabs

[0125]

[0126] Table 9: Nei's genetic distance and genetic similarity among the five populations of adult swimming crabs

[0127]

[0128] Note: The line above the diagonal is the genetic similarity coefficient, and the line below the diagonal is the genetic distance.

[0129] In summary, the present invention selected 19 pairs of highly polymorphic dibasic microsatellite primers, added fluorescent labels, and continuously optimized the reaction conditions. Four multiplex PCR systems were then assembled: one quadruplex PCR system and three quintuplex PCR systems. The multiplex PCR systems were used to perform paternity testing on 10 parentage samples. The results showed that the simulated identification rate using any two multiplex PCR systems was 100%, while the actual accuracy rate for paternity testing using three or more PCR systems exceeded 99%, demonstrating accurate identification capabilities.

Claims

1. A primer pair combination for multiplex PCR amplification of microsatellites of Portunus trituberculatus, characterized in that, The primer pair combination described above contains the following primer pairs: The SSR28-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:1 and the sequence of the downstream primer is SEQ ID NO:2; The SSR25-1 primer pair, where the sequence of the upstream primer is SEQ ID NO:3 and the sequence of the downstream primer is SEQ ID NO:4; The SSR114 primer pair, where the sequence of the upstream primer is SEQ ID NO:5 and the sequence of the downstream primer is SEQ ID NO:6; The SSR118 primer pair, where the sequence of the upstream primer is SEQ ID NO:7 and the sequence of the downstream primer is SEQ ID NO:

8.

2. A primer pair combination for multiplex PCR amplification of microsatellites of Portunus trituberculatus, characterized in that, The primer pair combination described above contains the following primer pairs: The SSR38-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:9 and the sequence of the downstream primer is SEQ ID NO:10; The SSR40-2 primer pair, where the sequence of the upstream primer is SEQ ID NO:11 and the sequence of the downstream primer is SEQ ID NO:12; The SSR21-2 primer pair, where the sequence of the upstream primer is SEQ ID NO:13 and the sequence of the downstream primer is SEQ ID NO:14; The SSR112 primer pair, where the sequence of the upstream primer is SEQ ID NO:15 and the sequence of the downstream primer is SEQ ID NO:16; The KX-61 primer pair, where the sequence of the upstream primer is SEQ ID NO:17 and the sequence of the downstream primer is SEQ ID NO:

18.

3. A primer pair combination for multiplex PCR amplification of microsatellites in Portunus trituberculatus, characterized in that, The primer pair combination described above contains the following primer pairs: The SSR 6-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:19 and the sequence of the downstream primer is SEQ ID NO:20; The SSR19-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:21 and the sequence of the downstream primer is SEQ ID NO:22; The SSR45-1 primer pair, where the sequence of the upstream primer is SEQ ID NO:23 and the sequence of the downstream primer is SEQ ID NO:24; The SSR1-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:25 and the sequence of the downstream primer is SEQ ID NO:26; The SSR117 primer pair, where the sequence of the upstream primer is SEQ ID NO:27 and the sequence of the downstream primer is SEQ ID NO:

28.

4. A primer pair combination for multiplex PCR amplification of microsatellites of Portunus trituberculatus, characterized in that, The primer pair combination described above contains the following primer pairs: The SSR34-1 primer pair, where the sequence of the upstream primer is SEQ ID NO:29 and the sequence of the downstream primer is SEQ ID NO:30; The SSR33-1 primer pair, where the sequence of the upstream primer is SEQ ID NO:31 and the sequence of the downstream primer is SEQ ID NO:32; The SSR42-3 primer pair, where the sequence of the upstream primer is SEQ ID NO:33 and the sequence of the downstream primer is SEQ ID NO:34; SSR19-2 primer pair, wherein the sequence of the upstream primer is SEQ ID NO:35 and the sequence of the downstream primer is SEQ ID NO:36; SSR119 primer pair, wherein the sequence of the upstream primer is SEQ ID NO:37 and the sequence of the downstream primer is SEQ ID NO:

38.

5. The primer pair combination according to any one of claims 1-4, characterized in that, The 5′ end or 3′ end of the primer pair carries a fluorescent label.

6. Use of the primer pair combination according to any one of claims 1-4 in detecting the genetic diversity of Portunus trituberculatus.

7. Use of the primer pair combination according to any one of claims 1-4 in the paternity testing of Portunus trituberculatus.

8. A method for paternity testing of swimming crabs, characterized in that, The method is to use the primer pair in the primer pair combination according to any one of claims 1-4 for identification.

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