Yangtze finless porpoise four-base repeat microsatellite molecular marker and construction method and application thereof

Abstract

The application discloses a Yangtze finless porpoise four-base repeat microsatellite molecular marker and a construction method and application thereof. The Yangtze finless porpoise four-base repeat microsatellite molecular marker comprises 19 microsatellite molecular markers, which are numbered as STR01-STR19, and the nucleotide sequences of the 19 Yangtze finless porpoise four-base repeat microsatellite molecular markers are shown as SEQ ID NO. 1-19. The four-base repeat microsatellite molecular marker is developed based on high-quality chromosome-level genome data and resequencing data of the Yangtze finless porpoise, and has stable amplification effect, high polymorphism and low genotyping error rate, and plays an important role in subsequent evaluation of genetic diversity of the Yangtze finless porpoise population, identification of kinship and prediction of population development.

Description

Technical Field

[0001] This invention belongs to the field of DNA molecular marker and genetic application technology, specifically relating to a tetrabase repeat microsatellite molecular marker for the Yangtze finless porpoise, its construction method and application. Background Technology

[0002] The Yangtze River is one of the world's most biodiverse rivers and a vital ecological barrier for my country. Since the late 20th century, rapid socio-economic development in the Yangtze River basin has intensified the pressures from human activities on water, leading to a continuous deterioration of the Yangtze River ecosystem and a severe situation for aquatic life conservation, including a sustained decline in the population of the Yangtze finless porpoise (Neophocaena asiae orientalis). A scientific survey in 2022 showed that the Yangtze finless porpoise population was approximately 1,249 individuals, an increase of 23.4% compared to 2017. Despite this historic rebound in population numbers, the population remains small, critically endangered, and the conservation situation remains dire. To effectively protect the germplasm resources of the Yangtze finless porpoise and formulate scientifically sound conservation strategies, a systematic analysis of its population's genetic diversity, genetic structure characteristics, and phylogenetic relationships is urgently needed.

[0003] Genetic markers are indispensable tools in the field of conservation genetics research. Mitochondrial DNA, due to its monolocution characteristic and maternal inheritance pattern, struggles to fully reflect the genetic characteristics of a population. In contrast, microsatellite DNA markers, with their abundant quantity, high polymorphic information content, co-dominant genetic characteristics, lower requirements for DNA quality, stable results, and strong reproducibility, are widely used in genetic diversity assessment, phylogenetic identification, phylogenetic geography, and evolutionary history research of endangered wildlife populations, thus greatly promoting the development of conservation genetics for endangered animals. Traditional microsatellite marker development methods, such as enrichment library screening, are often time-consuming, labor-intensive, and costly. High-quality chromosome-level genome data and resequencing data from different geographic populations of the Yangtze finless porpoise provide an opportunity to comprehensively mine microsatellite markers across the entire genome and screen for polymorphic sites among geographic populations. This method is superior to traditional methods in terms of effectiveness, accuracy, and cost-effectiveness. Currently, some researchers have developed microsatellite markers for the Yangtze finless porpoise based on amplified fragment length polymorphism (AFLP) methods. However, after examining these markers, it was found that (1) most microsatellite markers were dibase repeats, and there were obvious shadow bands in the amplification products, which led to deviations in the genotyping results; (2) some sites had low polymorphism, which led to unreliability of the results. Summary of the Invention

[0004] Technical problem solved: To address the above-mentioned technical problems, this invention provides a tetrabase repeat microsatellite molecular marker for the Yangtze finless porpoise, its construction method and application, which can effectively solve the shortcomings of the above-mentioned microsatellite molecular markers, such as unstable amplification effect, inaccurate genotyping results and low polymorphism.

[0005] Technical Solution: In a first aspect, this invention provides a tetra-base repeat microsatellite molecular marker for the Yangtze finless porpoise. The tetra-base repeat microsatellite molecular marker comprises 19 microsatellite molecular markers, numbered STR01-STR19. The nucleotide sequences of the 19 tetra-base repeat microsatellite molecular markers are shown in SEQ ID NO. 1-19. Specifically:

[0006] SEQ ID NO.1:

[0007] AAATATGCCTGTAGATGGATGGTTCGATGGTTGGATGTTTGGATACATGGAAGGATGGATGGGAGGGAGGAAAGGATGGAGAGAGAATAGATGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATGAGTGAATCAATCAATTAAATAGATAAATGCCATACCTTACTTGCATAAAGGAGTTCAACATCTT;

[0008] SEQ ID NO.2:

[0009] AACTAGAAATACACACTGACACCATCTAGAACTGAAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAATTGCCCATCTTGAAACATGAACATTGAATCTTGATCCTGATTGGAGGTAGAAAAGGATTTCTCCTGAAAA;

[0010] SEQ ID NO.3:

[0011] GCTGTCTCCTACCTTTTAATTATGATTATATAAATATAGATATAGACATCTATCTATCTATCTATCTATCTATCTATCTAATCTCCTCTAAGGTACCTGTAAGGTGCATGAGCCCTGAGCCAACACTCCTTTTGGGGATAATGCATGTTTCTGTGTTTCTTT;

[0012] SEQ ID NO.4:

[0013] GCAGAGAACAAAACCAAAAGGATGTGTATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAAAGAGAGTCAGTGAAAGTAAGAGGGGGCGGTAGGGAGAGGGAGAGATGGATTTTAAGGA;

[0014] SEQ ID NO.5:

[0015] AGAAAAACAGAACCAACAGGAGATAGGTGATAGATAAATAGATAGATAGGTAAGTAGATAGATGGATGGATGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATGGATAATGAAACCTTTGAGCCAACCTGGGCTCTTTGGCTCTGTTGCTCTGGGCAACTCTGTGTATGTTGGGAATATTAACATCCACCAACTAT;

[0016] SEQ ID NO.6:

[0017] TAAAAGAGACCAGACCATCTAGTCCAGAGGCTTAAATAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAAAAAAGTTCTTTTTCCACCTCTGAACTTCTTTTTGATGGAAAGCTGATCGTGTTAGGTCCAATATGTA;

[0018] SEQ ID NO.7:

[0019] CTTGACAGATTGGAAATTAAAAACAAAATCAACTAGGCCTTCCCTACAGATCATTTCAGAATTATTACCCTATCTATCTATCTATCTATCTCTATCTGTCTGATTTTCTCTAGATCTTTTTCCTACGTGAGAAACGACTTCAGCTAGCATTAAA;

[0020] SEQ ID NO.8:

[0021] ATCAATCAGTCAGCTACCTATCCACTCATCCATCTATTTATCATATTTATCATCTGTCATCTATCTATTTTATCTCCCTACCTTCTATATCTATCTCTCTCTTCTATCTCTCTCCCTTTATCTATCCATCATCTATCATCTGTCTGTCTATATCATCTATCATCTATTATCTATCTATCTATCATCTATCTATCTTCTATCTATCTATCTTCTATCTATCTATCATCTATCTTCTATCTATCTATCTATCTATCTATCATCTATCATCTCATCTATCTCCCCCACTAGAATACAAGCTCTATAAGTTCATGACCCT;

[0022] SEQ ID NO.9:

[0023] GTTGAATGTTGGCCTAAGTAATGACCAGAAATCTCACTGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCAGGCTGGTAGGCAGGCTTCCAAGTCTCATGTGCCTCTGATTT;

[0024] SEQ ID NO.10:

[0025] CTACTGAGCCTGTTCTCTAGAGCCCGTGAGCCATAACTACCGAAGCTTATGCGCCTAGAGCCCATGCTCCGCAACAAGCCACCACAATAAGTAGCCCACGCACTGCAGTGAAGAGTAGCCCCTGTTTGCCGCAACTAGAGAAAGCCCGCATGCAGCAACGAAGACCCAACACAGCCAAAAATAAAGAAAGAAAGAAAGAAAGAAAGAAAAGAAAACAAAAAGGGTCAACTGTACTTTTACAGAAGAATAAATTATCATTTATATTCAGAACATGTATGTAACTGATATATCTGCCTGGTTATATAGAAAGGTAAGATTTCTTTCTGCC;

[0026] SEQ ID NO.11:

[0027] GCTTTCTTGGTTTTACAGATTACAGATTGCAAATTGTGATACTTCTCAACCTCATAATTGGTGAGTCAATTCCTATAATAAATCTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCATCTCTCCTTATATATTTCCTAATGGTTCTGTTTCTCTG;

[0028] SEQ ID NO.12:

[0029] TCATTAACCTGCATAAGGGTCCTGTGATGTAGGGATTAACCTCCCAATTTTATGTATGTATGTATGTATGTATGTATGTATGTATGTATGTATGGCTGCACTGGGTCCTCGTTGCTGTGCACGAGCTTTCTCTAGTTGCGGCGAGTGGGGGCTACTCTTCGTTGTGGTGCATG;

[0030] SEQ ID NO.13:

[0031] TGTGTGTATTAGTCAGTGTTCTCCAGAGAAACAGAACCAATAGGAAATTAGATAATAGATAGATAGATAGATAGGTAGATAGATAGATTAGATTTATTACAAGAATTGGCTCACAT;

[0032] SEQ ID NO.14:

[0033] CTCTGTGGATAAACAATGAACTGTGAAAAAAACAATTAACTGTACTCTAAAATAGGTGATTTATATATGTGAAATATATCCCAGTAAAGCTGAGGAAAGAAGGAGGGAGGAAGGGAAGGAGGGAAGGAAGGAAAGAAGGAAGGAA GGAAGGACAGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAGGCAGGGAGGAAGGGAGGGAAGAAGGAAGGAATGAAAGATACAGATCTGAAGTGAAGTGTCTCACCCAGAATGGTAATTCTTCTCAGGGT;

[0034] SEQ ID NO.15:

[0035] TTCCTACATATTATGCACTACTGGGACAGTTCCTTCCTTCCTTCCATCTATCATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCAACTGGTTGGCCAAAAGTGCCTTCACTTTTTAAGTAAAAATAAAAGACACATTTTTCATTTTCACCA AGAACTTTATTGGACAACGTATTCACCCTTTTGTCCCACTACCATCTGCCGTTTTTCAGGCAACTTCATGATTCCATCTTCCCAAAACTTTTTATCTTTTTGAGCAAAGAACTGTTCCGGGTGCCTTTTACAGTTTTCCAAGGAACTGAAATCTTTTCCATTAAG;

[0036] SEQ ID NO.16:

[0037] TTAGGCACTGTTTTCAGTAGCATTGCTATGGATAGTAATGTTTTCTTTCTTTCTCTCTCTCTTTCTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTATTTCTAATTCCAGTTTTTCTAATTATTTCATATGAAATGAATAATCCCATGGTCCAATCCCCAGTCATCCATAGAAGGCTCCTGACTCACTTCTC;

[0038] SEQ ID NO.17:

[0039] CTGTGAAGGTACAGCATAAATGTGTGAAAATAAAATATCTCCTGACCTCGCCATCAGAGACATAGATTTTATTTTTTTCTTAGGAGTGTCTGGTGAGTTTTCTTTCTTTCTTTCCTTTCTTTCTCTTATTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCCTCTCTCTCTCTCTCTCTTTCTTTCTTCTTCTCCCTTTTTTTTTTGGTGGTGGTTGTCGCAGCTCAACGGGGCTTTACATTTAGGGAGTAGTCTG;

[0040] SEQ ID NO.18:

[0041] GTAGAATCACAGACACTCAGAGTGGTGAGGGCAGGAACTCTGCCAGGGAGCCCTGGGCCGGGGCAAGGATGTCCTTCCTGCTACCACCACAGGGAGGGAGGTAGGTAGGTAGGTAGGTAGGTAGGTAGGTAGGTAGGTAGCTGGCTGTCTCCTCTCATATCCTTGGCACATGAGGAGAAATCTGGGAGTATTATT;

[0042] SEQ ID NO.19:

[0043] GAACGCTCATTACCTAGGAACTTTAATTAATCTAGTTGAACTTAAGCTTAAAGAAATAATTGTTGGGGCTTCCCTGGTGGCGCAGTGGTTGGGAGTCCCGCCTGCCGATGCAGGGGACACGGGTTCGTGCCCCGGTCTGGGAGGATCCCACATGCCGCGGAGCGG CTGGGCTCGTGAGCCATGGCCGCTGAGCCTGCGCGTCCGGAGCCTGTGCTCCACAACGGGAGAGGTCACAACAGTGAGAGGCCCGCGTACCGCAAAAAACAAACAAACAAACAAACAAACAAAAAAAAAAAGAAAATAATTGTTCAGTCCTACAGACAGAAAATG.

[0044] Secondly, this invention provides primer combinations for amplifying the tetrabase repeat microsatellite molecular markers of the Yangtze finless porpoise described in the first aspect. The primer combinations for amplifying 19 microsatellite molecular markers contain 19 pairs of primers, and their nucleotide sequences are shown in SEQ ID NO. 20-SEQ ID NO. 57. Specifically:

[0045] SEQ ID NO.20: AAATATGCCTGTAGATGGATGGTT;

[0046] SEQ ID NO.21: AAGATGTTGAACTCCTTTATGCAAG;

[0047] SEQ ID NO.22: AACTAGAAATACACACTGACACCAA;

[0048] SEQ ID NO.23:TTTTCAGGAGAAATCCTTTTCTAC;

[0049] SEQ ID NO.24:GCTGTCTCCTACCTTTTAATTATGA.

[0050] SEQ ID NO.25:AAAGAAACACAGAAACATGCATTAT;

[0051] SEQ ID NO.26:GCAGAAACAAACCAAAAGG;

[0052] SEQ ID NO.27:TCCTTAAAATCCATCTCTCCCTC.

[0053] SEQ ID NO.28:AGAAAACAGAACCAACAGGAGATA.

[0054] SEQ ID NO.29:ATAGTTGGTGGATGTTAATATTCCC;

[0055] SEQ ID NO.30:TAAAAGACCAGACCATCTAGTCC;

[0056] SEQ ID NO.31:TACATATTGGACCTAACAGATCAGC.

[0057] SEQ ID NO.32:CTTGACAGATTGGAAATTAAAAACA;

[0058] SEQ ID NO.33:TTTAATGCTAGCTGAAGTCGTTTC;

[0059] SEQ ID NO.34:ATCAATCAGTCAGCTACCTATCCAC;

[0060] SEQ ID NO.35:AGGGTCATGAACTTATAGAGCTTGT.

[0061] SEQ ID NO.36:GTTGAATGTTGGCCTAAGTAATGA;

[0062] SEQ ID NO.37:AAATCAGAGGCACATGAGACTTG;

[0063] SEQ ID NO.38:CTACTGAGCCTGTTCTCTAGAGCC;

[0064] SEQ ID NO.39:GGCAGAAAGAAATCTTACCTTTCTA;

[0065] SEQ ID NO.40:GCTTTCTTGGTTTTACAGATTACAG;

[0066] SEQ ID NO.41:CAGAGAAACAGAACCATTAGGAAA;

[0067] SEQ ID NO.42:TCATTAACCTGCATAAGGGTCC;

[0068] SEQ ID NO.43:CATGCACCACAACGAAGAGTAG;

[0069] SEQ ID NO.44:TGTGTGTATTAGTCAGTGTTCTCCA;

[0070] SEQ ID NO.45:ATGTGAGCCAATTCTTGTAATAAAT;

[0071] SEQ ID NO.46:CTCTGTGGATAAACAATGAACTGTG;

[0072] SEQ ID NO.47:ACCCTGAGAAGAATTACCATTCTG;

[0073] SEQ ID NO.48:TTCCTACATATTATGCACTACTGGG;

[0074] SEQ ID NO.49:CTTAATGGAAAAGATTTCAGTTCCT;

[0075] SEQ ID NO.50:TTAGGCACTGTTTTCAGTAGCATT;

[0076] SEQ ID NO.51:GAGAAGTGAGTCAGGAGCCTTCTA;

[0077] SEQ ID NO.52:CTGTGAAGGTACAGCATAAATGTGT;

[0078] SEQ ID NO.53:CAGACTACTCCCTAAATGTAAAGCC;

[0079] SEQ ID NO.54: GTAGAATCACAGACACTCAGAGTGG;

[0080] SEQ ID NO.55: AATAATACTCCCAGATTTCTCCTCA;

[0081] SEQ ID NO.56: GAACGCTCATTACCTAGGAACTTTA;

[0082] SEQ ID NO. 57: CATTTTCTGTCTGTAGGACTGAACA.

[0083] Thirdly, the present invention provides a method for constructing the tetrabase repeating microsatellite molecular marker of the Yangtze finless porpoise described in the first aspect, comprising the following steps:

[0084] Step 1: Using high-quality chromosome-level genome data of the Yangtze finless porpoise as a reference, the LobSTR software was used to systematically screen and analyze the polymorphic tetrabase repeat sequences in the resequencing data of 12 samples:

[0085] (1) Based on the genome data of the Yangtze finless porpoise, a LobSTR reference index was constructed using the lobstr_index.py script;

[0086] (2) After comparing the resequencing data with the reference genome data, the generated BAM file was sorted using SAMtools software;

[0087] (3) Based on the comparison file analysis of the resequencing data of 12 samples, the allelic genotypes of microsatellite loci were determined;

[0088] (4) Using VCFtools software, by setting the parameters "-min-alleles 3" and "-maf 0.1", tetrabase repeat microsatellite loci shared with an allele number greater than or equal to 3 were screened from all 12 resequencing data;

[0089] Step 2: Using a combination of PCR amplification and capillary electrophoresis, microsatellite loci with polymorphism and accurate genotyping were identified.

[0090] Preferably, the specific process of step one is as follows: 5291 tetrabase repeat microsatellite loci data were obtained through screening. Based on the principle of uniform distribution on chromosomes, 190 loci were randomly selected and primers were designed using Primer 5.0 software. The 5' end of the forward primer F of the primer combination was connected to the universal tag M13 (SEQ ID NO.58): 5'-TGTAAAACGACGGCCAGT-3'. 41 polymorphic microsatellite loci were initially screened and retained. The 5' ends of the corresponding forward primers of the 41 selected tags were modified with fluorescent groups (FAM, ROX, HEX) to resynthesize fluorescent primers.

[0091] Preferably, the combined PCR amplification and capillary electrophoresis technique in step two specifically involves using PCR technology to amplify the target sequence, then using capillary electrophoresis technology to separate alleles, and analyzing the electrophoretic pattern using GeneMapper 5.0 software to determine the microsatellite allele genotype of the sample.

[0092] Fourthly, this invention provides the application of the tetrabase repeat microsatellite molecular markers of the Yangtze finless porpoise described in the first aspect in the analysis of genetic diversity and identification of kinship among the Yangtze finless porpoise.

[0093] Preferably, the specific application process is as follows:

[0094] S1: Extracting genomic DNA from Yangtze finless porpoise samples;

[0095] S2: Using the genomic DNA of the Yangtze finless porpoise sample extracted in step S1 as a template, a PCR amplification reaction system was prepared according to 19 microsatellite molecular marker sites, and PCR amplification was performed using the corresponding primers of the primer combination.

[0096] S3: Perform capillary electrophoresis on the PCR amplification products obtained in step S2, read and analyze the fluorescence signal, and thus obtain the genotyping results of the Yangtze finless porpoise sample at 19 microsatellite molecular marker sites.

[0097] S4: Genetic diversity analysis was performed on the genotyping results of the Yangtze finless porpoise samples obtained in step S3 at 19 microsatellite molecular marker loci using Cervus 3.0 software;

[0098] S5: The genotyping results of the Yangtze finless porpoise samples obtained in step S3 at 19 microsatellite molecular marker loci were used to identify kinship using Coancestry V 1.0 software.

[0099] Furthermore, the PCR amplification reaction system in step S2 is 25 μl, specifically including: 1 μL DNA, 0.5 μL each of forward and reverse primers, 0.5 μL dNTP mix, 2.5 μL 10×Taq buffer, 0.2 μL Taq enzyme, and 19.8 μL ddH2O; the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles, and finally 72℃ extension for 10 min.

[0100] Beneficial effects: The microsatellite molecular markers provided by this invention are all derived from high-quality chromosome-level genomes and screened in conjunction with resequencing data, thus enabling a more comprehensive acquisition of highly variable microsatellite loci in the Yangtze finless porpoise genome. These characteristics make the microsatellite molecular marker loci provided by this invention not only applicable to population genetic analysis of the Yangtze finless porpoise and its closely related species, but also of high application value in phylogenetic identification, phylogenetic geography, and evolutionary history.

[0101] Unlike previous microsatellite molecular markers that were mainly based on di-base repeats, the tetra-base repeat microsatellite molecular marker sites provided in this invention are less likely to produce shadow bands due to strand slippage during PCR amplification, resulting in faster and more accurate data reading.

[0102] The microsatellite molecular marker sites provided by this invention have been verified by capillary electrophoresis and have obtained highly polymorphic markers, which have strong effectiveness and applicability. Attached Figure Description

[0103] Figure 1 This is a genotyping diagram of STR 01-02 sites in some samples according to the present invention;

[0104] Figure 2 This is a genotyping diagram of STR 03-04 sites in some samples according to the present invention;

[0105] Figure 3 This is a genotyping diagram of STR 05-06 sites in some samples according to the present invention;

[0106] Figure 4 This is a genotyping diagram of STR 07-08 sites in some samples according to the present invention;

[0107] Figure 5 This is a genotyping diagram of STR 09-10 sites in some samples according to the present invention;

[0108] Figure 6 This is a genotyping diagram of STR 11-12 sites in some samples according to the present invention;

[0109] Figure 7 This is a genotyping diagram of STR 13-14 sites in some samples according to the present invention;

[0110] Figure 8 This is a genotyping diagram of STR 15-16 sites in some samples according to the present invention;

[0111] Figure 9 This is a genotyping diagram of STR 17-18 sites in some samples according to the present invention;

[0112] Figure 10 This is a genotyping diagram of the STR 19 locus in some samples according to the present invention;

[0113] Figure 11 A phylogenetic diagram of 30 Yangtze finless porpoise samples. Detailed Implementation

[0114] The present invention will be described in detail below with reference to specific embodiments: Unless otherwise specified, the chemical reagents and experimental materials used in the embodiments can be obtained through commercial channels.

[0115] Example 1

[0116] S1. Obtaining tetrabase repeat microsatellite molecular markers for Yangtze finless porpoises

[0117] A large number of microsatellite markers were obtained using high-quality chromosome-level genome sequencing data of the Yangtze finless porpoise and resequencing data from three geographic populations. The specific screening steps included: constructing a LobSTR reference index using the lobstr_index.py script based on the Yangtze finless porpoise genome data; sorting the generated BAM files using SAMtools software after aligning the resequencing data with the reference genome data; determining the allele genotypes of microsatellite loci based on the alignment files of 12 resequencing data samples; and using VCFtools software, by setting the parameters "-min-alleles 3" and "-maf 0.1", screening out shared tetrabase repeat microsatellite loci with an allele count greater than or equal to 3 from all 12 resequencing data samples. A total of 5291 tetrabase repeat microsatellite loci were obtained through these screening criteria.

[0118] S2, Extraction of Yangtze Finless Porpoise Genomic DNA

[0119] Genomic DNA was extracted from Yangtze finless porpoise samples using the QIAGEN DNeasy Blood & Tissue kit.

[0120] S3. Screening of polymorphic microsatellite molecular markers

[0121] Based on the principle of uniform distribution on chromosomes, 190 sites were randomly selected from 5291 tetrabase repeat microsatellite loci as candidate microsatellite molecular markers. Primers were designed using Primer 5.0 software for the upstream and downstream 500 bp sequences of these 190 microsatellite loci. Each primer combination consisted of a forward primer F and a reverse primer R, with the 5' end of the forward primer F linked to the universal tag M13:5'-TGTAAAACGACGGCCAGT-3'. Genomic DNA was extracted from 15 Yangtze finless porpoise samples as templates, and PCR amplification was performed using 190 primer pairs. Forty-one polymorphic microsatellite loci were preliminarily screened and retained.

[0122] The 5' ends of the corresponding forward primers for the 41 selected polymorphic microsatellite loci were modified with fluorescent groups (FAM, ROX, HEX), and fluorescent primers were resynthesized. Using genomic DNA from Yangtze finless porpoise samples as templates, PCR amplification was performed using the modified forward primers and corresponding reverse primers. The amplification products were genotyped using an ABI 3730XL gene analyzer to further verify the primer polymorphism.

[0123] Nineteen tetrabase repeat microsatellite loci with stable amplification effects, high polymorphism, and low genotyping error rate were finally screened out. These microsatellite loci and their corresponding primers are shown in Table 1.

[0124] Table 1. Tetrabase repeat microsatellite marker sites and corresponding primer information for Yangtze finless porpoises.

[0125]

[0126]

[0127]

[0128] S4. Genetic diversity analysis based on the development of microsatellite molecular markers

[0129] Genomic DNA from 30 Yangtze finless porpoise samples was used as templates for genetic diversity detection. PCR amplification was performed using 19 pairs of fluorescently modified primers obtained from the screening process. The PCR amplification reaction volume was 25 μL, specifically including: 1 μL DNA, 0.5 μL each of forward and reverse primers, 0.5 μL dNTP mix, 2.5 μL 10×Taq buffer, 0.2 μL Taq enzyme, and 19.8 μL ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s (temperature decreased by 0.5℃ per cycle), 72℃ extension for 30 s, for 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles, and a final extension at 72℃ for 10 min. PCR amplification products were mixed with loading buffer HIDI (ABI) and internal control marker LIZ500 (ABI) and detected by capillary electrophoresis using a 3730XL (ABI) sequencer. The amplification products were genotyped using a 3730XL gene analyzer, and the microsatellite alleles of the samples were determined using Genemapper 5.0 software. Genetic diversity indicators such as allele count (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were calculated using Cervus 3.0 software.

[0130] The genetic diversity results of 30 Yangtze finless porpoise samples are shown in Table 2 below:

[0131] Table 2. Statistical analysis of genetic parameters of 19 microsatellite molecular markers in 30 Yangtze finless porpoise samples.

[0132]

[0133]

[0134] The average allele count at the 19 loci was 3.9, the average observed heterozygosity was 0.627, the average expected heterozygosity was 0.617, and the average polymorphism information content was 0.557, indicating that the 19 developed microsatellite molecular markers are highly polymorphic. Genotyping results of the 19 loci in some samples are shown below. Figure 1-10 As shown, the 19 microsatellite markers of this invention exhibited polymorphism in length in Yangtze finless porpoise samples, demonstrating high amplification stability and accurate genotyping. Therefore, these 19 microsatellite markers can be used for germplasm resource assessment of the Yangtze finless porpoise. Compared with the 21 microsatellite loci reported by Chen Minmin et al. in their article "Assessment of Inbreeding Status in the Tian'ezhou Ex-situ Conservation Finless Porpoise Population," the genetic diversity parameters in this invention are all higher.

[0135] S5. Phylogenetic identification based on the development of microsatellite molecular markers

[0136] The kinship coefficient r between individuals was calculated using the TrioML estimation method in Coancestry V.1.0 software. The kinship coefficient r between parent-child pairs or full siblings was 0.5, while the kinship coefficient r between half-siblings (same mother, different father) was 0.25. Kinship analysis of 30 Yangtze finless porpoise samples was conducted. Figure 11 As shown: a total of 4 mother-child relationships and 1 pair of half-sister relationships were identified.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tetrabase repeating microsatellite molecular marker for the Yangtze finless porpoise, characterized in that: The Yangtze finless porpoise tetrabase repeat microsatellite molecular markers comprise 19 microsatellite molecular markers, numbered STR01-STR19. The nucleotide sequences of the 19 Yangtze finless porpoise tetrabase repeat microsatellite molecular markers are shown in SEQ ID NO.1-19.

2. The primer combination for amplifying the tetrabase repeat microsatellite molecular marker of the Yangtze finless porpoise as described in claim 1, characterized in that: The primer combination for amplifying 19 microsatellite molecular markers contains 19 pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO.20-SEQ ID NO.

57.

3. The application of the tetrabase repeat microsatellite molecular marker of the Yangtze finless porpoise as described in claim 1 in the analysis of genetic diversity and identification of kinship of the Yangtze finless porpoise.

4. The application according to claim 3, characterized in that, The specific process is as follows: S1: Extracting genomic DNA from Yangtze finless porpoise samples; S2: Using the genomic DNA of the Yangtze finless porpoise sample extracted in step S1 as a template, a PCR amplification reaction system was prepared according to 19 microsatellite molecular marker sites, and PCR amplification was performed using the primer combination described in claim 2. S3: Perform capillary electrophoresis on the PCR amplification products obtained in step S2, read and analyze the fluorescence signal, and thus obtain the genotyping results of the Yangtze finless porpoise sample at 19 microsatellite molecular marker sites. S4: Genetic diversity analysis was performed on the genotyping results of the Yangtze finless porpoise samples obtained in step S3 at 19 microsatellite molecular marker loci using Cervus 3.0 software; S5: The genotyping results of the Yangtze finless porpoise samples obtained in step S3 at 19 microsatellite molecular marker loci were used to identify kinship using Coancestry V 1.0 software.

5. The application according to claim 4, characterized in that: The PCR amplification reaction system in step S2 is 25 μl, specifically including: 1 μL DNA, 0.5 μL each of forward and reverse primers, 0.5 μL dNTP mix, 2.5 μL 10× Taq buffer, 0.2 μL Taq enzyme, and 19.8 μL ddH2O; the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles, and finally 72℃ extension for 10 min.

Images