Primer combination for paternity test of holothuria leucospilota based on microsatellite and application of primer combination
By designing a microsatellite-based primer combination and fluorescent capillary electrophoresis technology for parentage identification of sea cucumbers, we have achieved efficient and accurate identification of parentage in sea cucumbers, overcoming the shortcomings of traditional labeling methods and enabling accurate evaluation of the propagation and release effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-04-03
AI Technical Summary
Existing technologies lack efficient methods for paternity testing, and traditional physical marking methods are not applicable to sea cucumber juveniles, making it difficult to evaluate the effectiveness of the propagation and release of *Gnaphalium affine*.
A set of primer combinations for parentage identification of sea cucumbers with white feet based on microsatellites was designed. Genotyping was performed using fluorescent capillary electrophoresis. Parentage identification of sea cucumbers with white feet was carried out using 8 pairs of fluorescently labeled microsatellite primers. Parentage was determined by PCR amplification and genotyping software analysis.
This method enables efficient and accurate identification of parentage in sea cucumbers with white feet, overcomes the shortcomings of traditional marking methods, accurately assesses the effects of stock enhancement and release, and improves the accuracy of identifying released individuals.
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Figure CN121780720A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a set of primer combinations for parentage identification of sea cucumbers with white feet based on microsatellites and their applications. Background Technology
[0002] Jade Foot Sea Cucumber ( Holothuria leucospilota It belongs to the phylum Echinodermata, class Holothuroidea, order Aspidochirotida, family Holothuriidae, and genus Holothuroidea. Holothuria The Chinese white-footed sea cucumber (Crassula ovata), widely distributed in the nearshore shallow waters of Fujian, Taiwan, Guangdong, Guangxi, Hainan, and the Xisha Islands in my country, is a common species of tropical and subtropical sea cucumber. Because it feeds on seabed sediment and organic detritus, it is known as the "seabed scavenger," playing a vital role in maintaining the stability of nearshore marine ecosystems and improving aquaculture environments. Furthermore, the Chinese white-footed sea cucumber has both medicinal and edible value, being rich in saponins, sea cucumber polysaccharides, and other active substances, possessing various effects such as antioxidant, anti-tumor, and immune-boosting properties. However, with increasing market demand, overfishing has led to a sharp decline in the natural resources of the Chinese white-footed sea cucumber. Artificial breeding and release are effective strategies to address the resource crisis of the Chinese white-footed sea cucumber. Releasing artificially bred juvenile Chinese white-footed sea cucumbers into the wild to restore their natural population helps maintain the acid-base balance of seawater and the stability of coral reef ecosystems.
[0003] Artificial breeding of *Ulva rubiginosa* has been successfully achieved, but the application of this species in stock enhancement and release is still in its early stages. Traditional physical marker methods are not suitable for marking sea cucumber juveniles. The evaluation of the effectiveness of *Ulva rubiginosa* stock enhancement and release requires the development of efficient parentage testing techniques. Microsatellite (SSR) markers have advantages such as ease of PCR amplification, adherence to Mendelian inheritance laws, and high polymorphism and stability, and are widely used in determining parentage, making them an effective means of accurately evaluating the effectiveness of stock enhancement and release. To date, however, microsatellite markers have not been applied to parentage testing of *Ulva rubiginosa*. Summary of the Invention
[0004] The purpose of this invention is to provide a set of primer combinations for parentage identification of *Sinocyclocheilus salina* based on microsatellites, combined with fluorescent capillary electrophoresis for genotyping, enabling parentage identification of *Sinocyclocheilus salina*. This method for parentage identification of *Sinocyclocheilus salina* using the above primer combinations is efficient and accurate, and can be applied to the evaluation of the effects of stock enhancement and release programs.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Based on the chromosome-level reference genome of *S. koreana* (NCBI: GCA_029531755.1), microsatellite sequencing and primer design were performed using the QDD3 program, resulting in 121 SSR loci and corresponding 121 SSR primer pairs. Body wall DNA was extracted from six *S. koreana* individuals using a high-efficiency animal genomic DNA extraction kit (Qingke). Temperature gradient PCR combined with agarose gel electrophoresis was used to screen for eight pairs of microsatellite primers that could stably amplify DNA and exhibited high polymorphism and specificity. The sequence information is as follows: The SSR3 primer pair has a forward primer of 5'-CCCAGTTATGGCCCAGAAGG-3' (shown in SEQ ID NO.1) and a reverse primer of 5'-CCCTCGAGTATGGGCTCATG-3' (shown in SEQ ID NO.2). The SSR13 primer pair has a forward primer of 5'-GGGTGAGCCATGTTACCTCA-3' (shown in SEQ ID NO.3) and a reverse primer of 5'-GGCTTTACGGTTTGAGTGCC-3' (shown in SEQ ID NO.4). The SSR14 primer pair has a forward primer of 5'-TGACTTGAGGACCTTGACCATC-3' (shown in SEQ ID NO.5) and a reverse primer of 5'-CGACAGATCAGAATGCCACG-3' (shown in SEQ ID NO.6). The SSR16 primer pair has a forward primer of 5'-ACACTGCAGGACATAGATGCC-3' (shown in SEQ ID NO.7) and a reverse primer of 5'-TCGTGAAAGAGGTTAAGCAGGG-3' (shown in SEQ ID NO.8). The SSR23 primer pair has a forward primer of 5'-GAAGGCGCTTCTCCTTGGAT-3' (shown in SEQ ID NO.9) and a reverse primer of 5'-TGCTGTGAGTGTATGCTGTGA-3' (shown in SEQ ID NO.10). The SSR31 primer pair has a forward primer of 5'-ACAACTGTGACGAAATAGGTATTTGT-3' (shown in SEQ ID NO.11) and a reverse primer of 5'-TCCTCAACCCTCATATGTAACGT-3' (shown in SEQ ID NO.12). The SSR48 primer pair has a forward primer of 5'-GGGACTTTAACGGAAGCAGT-3' (shown in SEQ ID NO.13) and a reverse primer of 5'-ACTTGAGCTACGAGGTGCAC-3' (shown in SEQ ID NO.14). And SSR50 primer pairs, consisting of at least one pair of the following: forward primer is 5'-GTGGCCTGTCTAGAGCATATT-3' (shown in SEQ ID NO.15) and reverse primer is 5'-GCTAGGACTTTCCAAGAGGGT-3' (shown in SEQ ID NO.16).
[0006] Subsequently, a fluorescent group (FAM, HEX, ROX, or TAMRA) was added to the 5' end of the forward primer, and DNA from 96 sea cucumbers was amplified by PCR. The amplified products were sequenced using an ABI-3730XL automated DNA analyzer, and allele lengths were read using GeneMapper 5.0 software. Genotyping data were analyzed using Cervus 3.0.7 to determine polymorphism information at each locus.
[0007] FAM was added to the 5' end of the forward primers of SSR3 and SSR16 primer pairs; HEX was added to the 5' end of the forward primers of SSR13, SSR23 and SSR50 primer pairs; ROX was added to the 5' end of the forward primers of SSR14 and SSR31 primer pairs; and TAMRA was added to the 5' end of the forward primers of SSR48 primer pair.
[0008] The PCR amplification system consisted of: 5 μL PCR mix (Tulugang Biotechnology), 0.25 μL forward primer (modified with fluorescent substance), 0.25 μL reverse primer, 3.5 μL DEPC water, and 1 μL template DNA (diluted to 10 ng / μL).
[0009] The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min; followed by 35 cycles of amplification, each cycle including denaturation at 95℃ for 15 s, optimal annealing temperature for 20 s, extension at 72℃ for 30 s; and finally extension at 72℃ for 5 min.
[0010] Furthermore, to confirm that these microsatellite markers can be used to determine parentage in *Sinocyclocheilus rubescens*, samples were collected from 6 parent *Sinocyclocheilus rubescens*, 7 offspring, and 13 adult *Sinocyclocheilus rubescens* unrelated to the offspring. DNA was extracted using the method described above, and PCR amplification was performed using six pairs of fluorescent primers (SSR3, SSR16, SSR23, SSR31, SSR48, and SSR50). After sequencing and genotyping of the products, parentage testing was performed using Cervus 3.0.7 software. At a 95% confidence level, this method achieved a 100% success rate in parentage testing for *Sinocyclocheilus rubescens*.
[0011] This invention provides a method for parentage testing of sea cucumbers with microsatellite markers: (1) Extraction of template DNA from parent-offspring sea cucumber; (2) PCR amplification was performed using the above primer combination (the forward primer has a fluorescent modification at the 5' end); (3) The products were sequenced using an ABI-3730XL fully automated DNA analyzer and genotyped using GeneMapper 5.0; (4) Use Cervus 3.0.7 to determine parentage based on allele length.
[0012] The present invention also includes the application of the above primer combination in the identification of parentage in sea cucumbers with white feet.
[0013] The present invention also includes the application of the above primer combination in the effect evaluation of the propagation and release of sea cucumbers.
[0014] The beneficial effects of this invention are as follows: This method, for the first time, achieves the identification of parentage in *Sinocyclocheilus rubescens* using genotyping data from eight pairs of microsatellite fluorescent primers, demonstrating high efficiency and accuracy. This method has significant application value in evaluating the effectiveness of *Sinocyclocheilus rubescens* propagation and release programs, solving the problem that traditional physical markers are unsuitable for marking sea cucumber juveniles, and enabling relatively accurate determination of whether recaptured sea cucumbers are released individuals. Attached Figure Description
[0015] Figure 1 Sequencing peak diagram of SSR3 loci in a single sample of sea cucumber with white feet; Figure 2 Sequencing peak diagram of SSR13 locus in a single sample of sea cucumber with white feet; Figure 3 Sequencing peak diagram of SSR14 locus in a single sample of sea cucumber with white feet; Figure 4 Sequencing peak diagram of SSR16 locus in a single sample of sea cucumber with white feet; Figure 5 Sequencing peak diagram of SSR23 site in a single sample of sea cucumber with white feet; Figure 6 Sequencing peak diagram of SSR31 site in a single sample of sea cucumber with white feet; Figure 7 Sequencing peak diagram of SSR48 site in a single sample of sea cucumber with white feet; Figure 8 This is a sequencing peak diagram of the SSR50 site in a single sample of *Salix rubra*. Detailed Implementation
[0016] Example 1 Screening of microsatellite marker primers for polymorphic sea cucumber (1) Locus Identification and Primer Design: Microsatellite sequencing was performed on the chromosome-level reference genome of *Saururus chinensis* (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_029531755.1 / ) using the QDD3 program. A total of 121 SSR loci were identified, including 2, 3, 4, and 5-base repeats, all with more than 20 repeats. Based on these loci, microsatellite marker primers were designed using QDD3.
[0017] (2) Site Screening: In this experiment, polymorphism screening of identified microsatellite sites was performed using six wild-caught sea cucumbers, and the optimal annealing temperature Tm for PCR was determined. First, body wall DNA was extracted from the six sea cucumbers using a high-efficiency animal genomic DNA extraction kit (Qingke) according to the manufacturer's instructions. The DNA concentration was diluted to 10 ng / μL with DEPC water and used as a template for temperature gradient PCR. The PCR reaction system consisted of: 10 μL PCR mix (Tulugang Bio), 0.4 μL forward primer, 0.4 μL reverse primer, 7.2 μL DEPC water, and 2 μL template DNA. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 35 cycles: 95℃ denaturation for 15 s, annealing temperature (gradient) for 20 s, 72℃ extension for 30 s; 72℃ extension for 5 min. The PCR products were verified by 1% agarose gel electrophoresis, and primers with high polymorphism and their corresponding optimal annealing temperatures were selected.
[0018] Table 1. Information on eight microsatellite marker primers for sea cucumber *Cynodon dactylon*. Example 2 Analysis of the characteristics of polymorphic microsatellite marker primers in sea cucumber *Cynodon dactylon* (1) Synthesis of fluorescently labeled primers: Based on the information of the 8 pairs of microsatellite labeled primers in Table 1, a fluorescent group (FAM, HEX, ROX or TAMRA) was added to the 5' end of each forward primer to synthesize fluorescently labeled primers. Specifically, the forward primers of SSR3 and SSR16 were added with the FAM group to the 5' end; the forward primers of SSR13, SSR23 and SSR50 were added with the HEX group to the 5' end; the forward primers of SSR14 and SSR31 were added with the ROX group to the 5' end; and the forward primers of SSR48 were added with the TAMRA group to the 5' end.
[0019] (2) DNA extraction and PCR amplification: Body wall DNA was extracted from 96 sea cucumbers using a high-efficiency animal genomic DNA extraction kit (Qingke). The DNA concentration was diluted to 10 ng / μL with DEPC water and used as a template for PCR amplification. The PCR reaction system and reaction procedure are shown in Table 2.
[0020] Table 2 PCR reaction system and reaction procedure (3) PCR product sequencing and genotyping: PCR products were detected by 1% agarose gel electrophoresis. The concentration of PCR products was initially determined based on the band brightness. PCR products of SSR3, SSR13, and SSR14 primer pairs, and SSR16, SSR31, SSR48, and SSR50 primer pairs were mixed separately and sequenced using an ABI-3730XL automated DNA analyzer (PCR products of SSR23 primer pair were sequenced separately). Allele lengths were read using GeneMapper 5.0 software.
[0021] (4) Microsatellite locus polymorphism and parental exclusion probability analysis: Cervus 3.0.7 software was used to analyze the allele frequencies of the genotyping data to determine the polymorphism information and exclusion probability of each locus. The polymorphism information of each microsatellite locus is shown in Table 3. The number of alleles at each locus ranged from 17 to 40; the observed heterozygosity ranged from 0.458 to 0.896; the polymorphism information content ranged from 0.763 to 0.929; and all 8 loci showed high polymorphism. The parental exclusion probability information of each microsatellite locus is shown in Table 4. When the genotypes of both parents were unknown, the non-parental exclusion probability of a single parent was 0.229 to 0.566; when the genotype of one parent was known, the non-parental exclusion probability of the other parent was 0.131 to 0.385; and when the genotypes of both parents were known, the non-parental exclusion probability of both parents was 0.026 to 0.185. When the parents are unknown, the cumulative exclusion rate of the 8 loci is 0.999, which can effectively exclude offspring who are not their parents.
[0022] Table 3. Polymorphism information of 8 microsatellite loci in sea cucumber *Sinocyclocheilus julibrissin*. Table 4. Parental exclusion probability information for 8 microsatellite loci in *Sinonovacula julibrissin*. Example 3 Testing the accuracy of paternity testing methods for sea cucumbers with white feet To determine whether microsatellite markers could accurately determine parentage in *Sinonovacula julibrissin*, body wall samples from 19 parent plants (including 6 true parents) and 7 offspring samples were collected and preserved in anhydrous ethanol. DNA was extracted using the above method, and PCR amplification was performed using six pairs of fluorescent primers: SSR3, SSR16, SSR23, SSR31, SSR48, and SSR50. PCR products were detected by 1% agarose gel electrophoresis, and the concentration of PCR products was initially determined based on band brightness. The PCR products from SSR3, SSR23, and SSR31 primer pairs, and SSR16, SSR31, and SSR50 primer pairs were then mixed separately and sequenced using an ABI-3730XL automated DNA analyzer. Genotyping was performed using GeneMapper 5.0 software. Parentage determination was performed using Cervus 3.0.7. The identification results are shown in Table 5. When both parents are unknown, the cumulative exclusion probability of parents for the six microsatellite loci is 0.998. All seven offspring individuals were able to quasi-identify their parents. At a 95% confidence level, this method can achieve a 100% success rate in paternity testing for *Sinonovacula julibrissin*.
[0023] Table 5. Paternity test results of 7 offspring samples
Claims
1. A primer combination for parentage testing of *Sinonovacula julibrissin* based on microsatellites, characterized in that, The sequence information is as follows: The SSR3 primer pair has the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.
2. The SSR13 primer pair has the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.
4. The SSR14 primer pair has the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.
6. The SSR16 primer pair has the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.
8. The SSR23 primer pair has the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.
10. The SSR31 primer pair has the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.
12. The SSR48 primer pair has the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.
14. And the SSR50 primer pair, the forward primer of which is shown in SEQ ID NO.15 and the reverse primer of which is shown in SEQ ID NO.16, consists of at least one pair.
2. The primer combination for microsatellite-based parentage testing of *Salix rubra* as described in claim 1, characterized in that: FAM was added to the 5' end of the forward primers of SSR3 and SSR16 primer pairs; HEX was added to the 5' end of the forward primers of SSR13, SSR23 and SSR50 primer pairs; ROX was added to the 5' end of the forward primers of SSR14 and SSR31 primer pairs. TAMRA was added to the 5' end of the forward primer of the SSR48 primer pair.
3. The primer combination for microsatellite-based parentage identification of sea cucumber as described in claim 1 or 2 is applied to the identification of parentage in sea cucumber.
4. The application of the primer combination for microsatellite-based parentage identification of *Sinocyclocheilus edulis* as described in claim 1 or 2 in the evaluation of the effect of *Sinocyclocheilus edulis* propagation and release.
5. A method for parentage testing of *Sinonovacula julibrissin* based on microsatellite markers, characterized in that... Includes the following steps: (1) Extraction of template DNA from parent-offspring sea cucumber; (2) PCR amplification was performed using the primer combination described in claim 2; (3) The products were sequenced using an ABI-3730XL fully automated DNA analyzer and genotyped using GeneMapper 5.0; (4) Use Cervus 3.0.7 to determine parentage based on allele length.