Earthworm nuclear gene molecular marker primer, molecular marker and molecular marker database

Abstract

The invention provides an earthworm nuclear gene molecular marker primer, a molecular marker and a molecular marker database, and belongs to the technical field of gene engineering, and the earthworm nuclear gene molecular marker primer comprises one or more of primers as shown in SEQ ID NO.1-SEQ ID NO.40. On the basis of genome data of five earthworms and one sea worm, 10 general type nuclear gene molecular markers of the earthworm are successfully developed and designed, and an earthworm general type nuclear gene molecular marker toolkit is formed. The molecular marker which is universal for various families is developed for the earthworm nuclear gene for the first time. The molecular marker primers are widely tested in a high-span classification order group, the performance is excellent, and the average success rate of PCR (Polymerase Chain Reaction) data is 80%. In tests of 12 types of earthworms, the molecular markers not only have strong variation signals, can be used for defining earthworm species, but also have excellent performance in construction of earthworm phylogenetic trees and biogeography analysis.

Description

Technical Field

[0001] The present invention relates to the acquisition of earthworm nuclear gene molecular markers and a molecular marker database, and in particular to an earthworm nuclear gene molecular marker primer, a molecular marker and a molecular marker database, belonging to the technical field of genetic engineering. Background Art

[0002] Earthworms, as important members of the class Oligochaeta in the phylum Annelida, are widely distributed worldwide and play a key role in ecosystems. Earthworms are not only core players in nutrient cycling and biodiversity maintenance, but are also known as "ecosystem engineers" for their multifunctionality within ecosystems. However, despite their irreplaceable importance in global ecosystems, their species identification faces numerous challenges. Currently, the estimated number of earthworm species worldwide is as high as 30,000, but only approximately 6,000 have been identified. Furthermore, the phylogeny and evolutionary history of earthworms remain unclear. The high degree of conservation of earthworm morphology is one of the main obstacles to species identification. Therefore, using genetic molecular data to conduct species classification and identification of earthworms, as well as in-depth analysis of their genetic lineages and biogeographic distribution, is of great significance for the conservation of earthworm species resources and the exploration of their ecological value.

[0003] In recent years, molecular marker technology has made significant progress in biological classification and phylogenetic research. For example, Thomson et al. (2008) designed 96 nuclear protein-coding molecular markers for reptiles, turtles, and screened out 11 markers that can be used for phylogenetic analysis; Li et al. (2010) developed a database of 210 non-coding candidate genes based on five bony fish genome data and successfully screened out five intronic molecular markers for phylogenetic studies of bony fish; Chenuil et al. (2010) designed 52 primer pairs for 29 genes in metazoans and ultimately obtained five intronic molecular markers for phylogenetic studies; Shen et al. (2012) developed a toolkit containing 102 nuclear protein-coding genes, which can quickly and accurately obtain target fragments through PCR methods and has been successfully applied to phylogenetic studies of salamander groups; Che et al. (2017) developed 95 universal nuclear protein-coding molecular markers for Coleoptera, which have played an important role in phylogenetic studies of Carabidae and Coccinellidae (Zhang et al. et al., 2018); Li et al. (2017) developed 96 universal intronic molecular markers for the snake suborder for species delimitation and phylogenetic analysis of snake groups; Mou et al. (2022) developed the FishPIE toolkit consisting of 82 nuclear gene molecular markers for ray-finned fishes and applied it to the phylogenetic analysis of 2023 fish species.

[0004] The successful cases of these studies show that molecular marker technology has great potential in biological classification and phylogenetic research, and also provides new ideas and methods for earthworm species identification and phylogenetic research.

[0005] However, current molecular marker primer set development and design are mostly based on vertebrates or arthropods. For earthworms, a key component of annelids, commonly used molecular markers are limited to COX1 (cytochrome c oxidase subunit I), 16S rRNA (16S ribosomal RNA gene), 28S rRNA (28S ribosomal RNA gene), and ITS (internal transcribed spacer). The first two of these molecular markers are mitochondrial genes. While mitochondrial genes offer advantages such as rich interspecific variation and relatively easy amplification, they are maternally inherited and cannot provide parental evolutionary signals. The latter two are nuclear gene markers. While they can provide parental evolutionary signals, their sequence variation is low, which can sometimes be insufficient. Furthermore, for radiating evolutionary groups, these markers lack sufficient variability, making them inadequate for species delineation and phylogenetic studies. Therefore, there is still a gap in the development of earthworm nuclear gene molecular markers, and there is an urgent need to develop new, universal earthworm nuclear gene molecular markers. Summary of the Invention

[0006] To address the above problems, the present invention provides an earthworm nuclear gene molecular marker primer, molecular marker and molecular marker database to facilitate rapid germplasm identification and analysis of earthworm evolutionary origin relationships and other aspects of the research.

[0007] To achieve the above object, the technical solution of the present invention is: a universal nuclear gene molecular marker primer for earthworms, including one or more primers shown as SEQ ID NO.1 to SEQ ID NO.40.

[0008] Further, it includes one or more primer pairs among EW01-EW10, wherein primer pair EW01 is shown as SEQ ID NO.1 to SEQ ID NO.4; primer pair EW02 is shown as SEQ ID NO.5 to SEQ ID NO.8; primer pair EW03 is shown as SEQ ID NO.9 to SEQ ID NO.12; primer pair EW04 is shown as SEQ ID NO.13 to SEQ ID NO.16; primer pair EW05 is shown as SEQ ID NO.17 to SEQ ID NO.20; primer pair EW06 is shown as SEQ ID NO.21 to SEQ ID NO.24; primer pair EW07 is shown as SEQ ID NO.25 to SEQ ID NO.28; primer pair EW08 is shown as SEQ ID NO.29 to SEQ ID NO.32; primer pair EW09 is shown as SEQ ID NO.33 to SEQ ID NO.36; and primer pair EW10 is shown as SEQ ID NO.37 to SEQ ID NO.40.

[0009] A universal nuclear gene molecular marker for earthworms is formed by amplifying the above-mentioned molecular marker primers.

[0010] A universal earthworm nuclear gene molecular marker database includes the above-mentioned universal earthworm nuclear gene molecular marker.

[0011] The use of the above-mentioned earthworm universal nuclear gene molecular marker primers, the above-mentioned earthworm universal nuclear gene molecular markers, and the above-mentioned earthworm universal nuclear gene molecular marker database in preparing reagents for identifying earthworm germplasm resources and categories.

[0012] A method for identifying earthworm species comprises the following steps:

[0013] Step S1, obtaining DNA of the earthworm to be identified;

[0014] Step S2, performing PCR amplification using the above primers;

[0015] Step S3, performing high-throughput sequencing on the PCR amplification product obtained in step S2 and comparing it with the above-mentioned earthworm universal nuclear gene molecular marker database, obtaining the species information of the earthworm to be identified based on the molecular markers in the molecular marker database that match the earthworm to be identified and the phylogenetic tree established based on the molecular marker database.

[0016] A kit comprises the above-mentioned earthworm universal nuclear gene molecular marker and / or the above-mentioned earthworm universal nuclear gene molecular marker primer.

[0017] Furthermore, it also includes reagents required for the PCR amplification system of the above-mentioned earthworm universal nuclear gene molecular marker primers.

[0018] A phylogenetic model is constructed using the earthworm universal nuclear gene molecular marker primers, the earthworm universal nuclear gene molecular markers and / or the earthworm universal nuclear gene molecular marker database.

[0019] Furthermore, phylogenetic trees are included.

[0020] The beneficial effects of the earthworm nuclear gene molecular marker primers, molecular markers and molecular marker database of the present invention are:

[0021] Based on the genomic data of 5 earthworm species and 1 marine worm species, the present invention successfully developed and designed 10 universal nuclear gene molecular markers for Oligochaeta (earthworms), constituting a universal nuclear gene molecular marker toolkit for earthworms. This is the first time that molecular markers universal to all families have been developed for earthworm nuclear genes. These molecular marker primers have been extensively tested in high-span taxonomic groups and performed well, with an average PCR data success rate of 80%. In tests on 12 earthworm species, these molecular markers not only have strong variation signals that can be used to define earthworm species, but also perform well in constructing earthworm phylogenetic trees and biogeographic analyses.

[0022] The earthworm nuclear gene molecular marker developed by the present invention has wide applicability and can be used in earthworm species of different families. It has good versatility and a high amplification success rate. Its sequence can not only be used for wild earthworm species identification, but can also be widely used in soil ecological surveys and beneficial animal diversity protection, providing strong support for the scientific management and ecological protection of earthworm resources.

[0023] The present invention fills the gap in earthworm nuclear gene molecular markers, provides a powerful tool for earthworm species identification, phylogenetic research and biogeographic analysis, and is expected to promote the development of earthworm taxonomy, phylogenetics and evolutionary biology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Figure 1 The following are examples of electrophoretic patterns of PCR amplification products of molecular markers designed in the embodiments of the present invention in 12 species. The upper figure shows the electrophoretic pattern of molecular marker 02 amplified using primer pair EW02 in 12 species, and the lower figure shows the electrophoretic pattern of molecular marker 04 amplified using primer pair EW06 in 12 species. The lengths of DNAmakers in the figures, from top to bottom, are 2000 bp, 1000 bp, 750 bp, 500 bp, 200 bp, and 100 bp, respectively.

[0026] Figure 2 is a genetic distance map of the molecular markers newly developed in the embodiments of the present invention;

[0027] Figure 3 This is a phylogenetic relationship diagram of earthworms constructed using newly developed molecular markers in an embodiment of the present invention. The numbers near the nodes in the diagram are support rates.

[0028] Figure 4-1 to Figure 4-21 This is an embodiment of the present invention Figure 1 The gene sequence corresponding to the electrophoretic bands in the middle. DETAILED DESCRIPTION

[0029] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Based on the genome data of five earthworm species and one marine worm species, the present invention successfully developed and designed 10 universal nuclear gene molecular markers for Oligochaeta (earthworms), forming a universal nuclear gene molecular marker tool kit for earthworms.

[0031] Example 1 Development of universal nuclear gene molecular markers for earthworms

[0032] 1. Screening process of molecular marker toolkit

[0033] The present invention uses the genomes of five oligochaete earthworms (including Metaphirevulgaris, Eiseniafetida, Aporrectodea caliginosa, Bimastos eiseni, Lumbricus terrestris) published in the NCBI database and one polychaete sea worm Capitella Based on the genome data of the earthworm (E. teleta), molecular marker screening was carried out. Genes suitable for earthworm molecular markers must meet the following criteria: First, the gene must be a single-copy homolog in multiple genomes. This step was implemented by BLAST alignment, with each molecular marker having only one hit in each genome. These markers were orthologous to single-copy genes in six different genomes (NCBI accession numbers are shown in Table 1). Finally, candidate genes suitable for primer design were selected by fine-snipping and screening the single-copy gene sequences, and amplification primers were designed. The principle of fine-snipping and screening was to ensure that at least two regions of conserved sequence of 8 amino acids were present at both ends of the molecular marker. The outer region was used for the first round of primer design, and the inner conserved region was used for the second round of primer design. A certain degree of degeneracy (degeneracy ranged from 0 to 8192 bases) was designed. After rigorous screening, the sequence length was limited to 350-1200 bases. Ultimately, 10 candidate molecular markers were identified.

[0034] The degeneracy of a primer refers to the fact that the primer can bind to multiple different template sequences due to the presence of multiple possible bases (i.e., degenerate bases) at certain positions in the primer sequence. The degeneracy is calculated as follows:

[0035] 1. Determine the location and type of degenerate bases

[0036] Find the degenerate bases and their corresponding base combinations contained in the primer sequence. For example:

[0037] R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T, D=A / G / T, V=A / C / G, N=A / C / G / T

[0038] 2. Calculate the number of possible combinations for each degenerate base

[0039] Based on the definition of degenerate bases, determine the number of possible bases at each degenerate base position. For example:

[0040] R = 2 possibilities (A or G), Y = 2 possibilities (C or T), H = 3 possibilities (A, C, or T), N = 4 possibilities (A, C, G, or T)

[0041] 3. Calculate the total degeneracy

[0042] Multiply the number of possible combinations of all degenerate base positions to obtain the total degeneracy of the primer. For example, if the primer sequence is CTTGGAGTBAARACTGTG, where there are three possible combinations of B (C / T / G) and two possible combinations of R (G / A), the degeneracy is 3 × 2 = 6, meaning that this primer can bind to six different template sequences.

[0043] Example

[0044] Assume the primer sequence is CTTGGAGTBAARACTGTG:

[0045] B(C / T / G): 3 possibilities, R(G / A): 2 possibilities, total degeneracy = 3×2 = 6

[0046] Table 1 NCBI accession numbers of the six annelid genomes used for molecular marker screening

[0047] Latin name Chinese name Data accession number Metaphirevulgaris Common earthworm GCA_018105865 Eiseniafetida Eisenia fetida GCA_003999395 Aporrectodeacaliginosa Back dark current earthworm GCA_020284085 Bimastoseiseni Aisheng Diplethora GCA_959347315 Lumbricus terrestris Earthworm GCA_949752735 Capitellateleta Sea worms GCA_000328365

[0048] 2. Composition of the Molecular Marker Toolkit

[0049] To improve amplification efficiency, the present invention adopts a nested PCR amplification strategy, i.e., two rounds of primers are designed: the upstream and downstream primers in the first round are used for enrichment of the target fragment, and the upstream and downstream primers in the second round are used for specific amplification of the target fragment. To ensure that the primers can be successfully amplified in all groups of earthworms, we designed two rounds of primers with degenerate primers of 20bp and 23bp, respectively. Based on the above strategy, we designed primers for 10 candidate molecular markers (Table 2) and commissioned Beijing Tianyi Huiyuan Biotechnology Co., Ltd. to complete the primer synthesis. The 40 primers designed for these 10 molecular markers constitute the universal molecular marker tool kit for earthworm nuclear genes. In Table 2, earthworm can also be abbreviated as EW.

[0050] Table 2 Universal earthworm molecular marker toolkit developed and designed by the present invention

[0051]

[0052]

[0053] Example 2

[0054] In order to verify the performance of the newly developed earthworm molecular marker toolkit, the present invention selected 12 oligochaete earthworm species covering multiple families and genera for PCR amplification experiments to evaluate the amplification effect of the primers. The test species included four earthworm species from the family Lumbricidae (Eisenia nordenskioldi, Octolasion tyrtaeum, Dendrobaena octaedra, and Bimastos parvus); three earthworm species from the family Megalobrachiidae (Metaphire changbaimontis, Metaphire tchiliensis, and Amynthashupeiensis); one earthworm species from the family Glossopsidae (Pontoscolex corethrurus); and three earthworm species from the family Streptogasteridae (Drawida ghilarovi, Drawida japonica, Drawidagisti, and Desmogastersinensis). Twelve earthworm species were collected from the wild and immediately stored at -20°C.

[0055] DNA extraction was performed using sterile scissors to cut the tail tissue and the whole genome extraction kit (TIANamp Genomic DNA Kit; TIANGEN Inc., Beijing, China) produced by Beijing Tiangen Biotechnology Co., Ltd. was used. The specific steps were strictly followed according to the kit instructions.

[0056] The reaction system and program settings for PCR amplification are detailed in the table below. During the experimental operation, DNA template, primers, dNTPs, reaction enzyme and PCR buffer were added in sequence. The template used in the first round of PCR was genomic DNA diluted to 30 ng / μL, while the template for the second round of PCR was the product enriched in the first round of PCR (Table 3, Table 4). After the second round of PCR was completed, 1% agarose gel electrophoresis was used to detect the amplification of the target fragment, and 1 μL of the reaction product was taken from each well for electrophoresis analysis. The results of electrophoresis detection showed that the PCR success rate of each molecular marker was higher than 80% (Table 5, Figure 1 ), indicating that the molecular marker toolkit developed in the present invention is highly efficient and reliable in earthworm species identification. Figure 1 The numbers 1-12 in the table correspond one to one with the earthworm species listed in Table 5.

[0057] Table 3 Reaction system and procedure of PCR process

[0058]

[0059] Table 4 PCR reaction program (first round PCR annealing temperature is 45°C, second round PCR annealing temperature is 45°C)

[0060]

[0061] Table 5. Amplification results and sequence lengths of 10 molecular markers in 12 earthworm specimens (unit: bp, "no" indicates that no valid sequence was assembled)

[0062]

[0063] The primers used to amplify 12 earthworm specimens using molecular marker EW01 were the aforementioned EW01 primer pair; the primers used to amplify 12 earthworm specimens using molecular marker EW02 were the aforementioned EW02 primer pair, and so on. The base composition of the sequences of the earthworm specimens amplified using each molecular marker primer pair is represented by 1-1, 1-2, ..., 1-12, 2-1, 2-1, ..., 2-12, ..., where 1-1 is the base composition of the sequence amplified using primer pair EW01 for Eisenia nordenskioldi, 1-2 is the base composition of the sequence amplified using primer pair EW01 for Octolasion tyrtaeum, and so on.

[0064] in accordance with Figure 1 The electrophoresis bands are shown, and the gene sequences corresponding to each band are shown as an example. Figure 4-1 to Figure 4-21 shown.

[0065] The earthworm universal nuclear gene molecular marker is formed by amplification of the above molecular marker primer pair, and the earthworm universal nuclear gene molecular marker database includes the above earthworm universal nuclear gene molecular marker.

[0066] Combined with the above table, we can see that among the 12 species, the number of newly designed molecular markers successfully amplified is 9-12, and the average data success rate is as high as 80%. Figure 1 , Table 5)

[0067] Example 3

[0068] 1. Phylogenetic evaluation of molecular marker accuracy

[0069] On the basis of Example 2, the accuracy of the generated molecular markers was evaluated based on the phylogenetic relationships of known earthworm species. First, a phylogenetic tree of each molecular marker was constructed. Specifically, MAFFT software was used to perform sequence alignment on each gene (each gene sequence amplified in Example 2), and RAxML v8.0 software was used to perform maximum likelihood tree construction on the aligned data. The model was GTR+Γ+I, and the support rate of each node was estimated by 500 rapid bootstraps.

[0070] Calculations revealed that the newly developed molecular markers have a high genetic variation, measured by the p-distance indicator. P-distance is a simple method for measuring the difference between two sequences. Its definition and calculation method are as follows: p-distance is the ratio of different nucleotide sites between two sequences. It is obtained by calculating the number of sites where the two sequences differ in the compared nucleotide sites and then dividing it by the total number of sites compared. In the 12 test samples, the average genetic distances of the 10 molecular markers at the three taxonomic levels (family, genus, and species) were 0.26, 0.12, and 0.15, respectively. Figure 2 ).

[0071] 2. Using the molecular marker primers and molecular markers of the present invention to construct phylogenetic relationships at the earthworm species level:

[0072] The newly designed molecular markers showed good amplification stability and data consistency in different species, and could provide sufficient signal for the analysis of phylogenetic relationships. A phylogenetic tree was constructed for 12 species using 10 molecular markers. The topology of the phylogenetic tree was stable, with 90% node support (Bootstrap>96), and the phylogenetic relationships were accurate, reflecting the phylogenetic relationships at the earthworm species level, such as between species, between genera, and between families ( Figure 3 ).

[0073] Example 4

[0074] Based on Example 2, high-throughput library construction and sequencing were performed: In order to perform high-throughput sequencing, the present invention mixed the PCR products of the same species, and then fragmented the PCR products using NEBNext dsDNAFragmentase enzyme so that their lengths were distributed between 200-800bp. The fragmentation products of different species were connected with unique tags (index) to distinguish them in subsequent analysis. The fragmentation products of all species were mixed to construct a sequencing library, and Novogene was commissioned to perform high-throughput sequencing. The data obtained by sequencing were 150bp bidirectional sequencing reads. The reads were sorted by unique tags, and the fragments were spliced using the splicing software Trinity. Finally, the reference sequences were compared using BLAST to identify specific molecular marker names.

[0075] Example 5

[0076] The identification of earthworm species and species definition using the molecular marker primers and molecular markers of the present invention includes the following steps:

[0077] 1) DNA extraction: A small piece of tissue was selected from the earthworm specimen, and whole-genome DNA was extracted using a DNA extraction kit.

[0078] 2) PCR amplification: Two rounds of nested PCR amplification were performed using the 10 molecular marker primers designed by the present invention.

[0079] 3) Sequencing of amplified products: The products from the second round of amplification are sent to a biotechnology company for Sanger or high-throughput sequencing (second-generation high-throughput sequencing is recommended, as high-throughput sequencing can obtain accurate molecular data even for faint bands) to obtain the base sequence of the molecular marker.

[0080] 4) The determined sequence was compared with the reference sequence (the reference sequence in Example 4) using BLAST software.

[0081] 5) For undescribed new species, species delimitation analysis can be performed using software such as BPP and ASAP.

[0082] In summary, the newly designed molecular markers demonstrated excellent amplification success rate, evolutionary signal strength, and stability, demonstrating their high application value. This demonstrates that the newly designed molecular marker primers in this patented invention excel in all aspects and are suitable for species delimitation, phylogenetic analysis, and biogeographic analysis, making them important molecular tools in soil ecological surveys.

[0083] This invention fills the gap in earthworm nuclear gene markers. Compared to existing mitochondrial gene markers (such as COX1 and 16S), the universal nuclear gene markers of this invention can reflect a more comprehensive genetic diversity of earthworm populations. Nuclear gene markers can provide the genetic history of both parents, thus avoiding the uniparental bias caused by maternal inheritance of mitochondrial genes.

[0084] Compared to existing technologies, the primers designed in this invention utilize nested PCR amplification, resulting in a high amplification success rate and stable bands. These 10 molecular markers carry more parsimonious loci than mitochondrial markers and can be used for genetic analysis within, between, and between populations, improving identification and delineation of earthworm species and phylogenetic relationships.

[0085] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

Claims

1. A universal nuclear gene molecular marker primer for earthworms, characterized in that: It includes one or more primers shown in SEQ ID NO.1 to SEQ ID NO.

40.

2. The universal nuclear gene molecular marker primer for earthworms according to claim 1, characterized in that: The invention comprises one or more primer pairs selected from EW01 to EW10, wherein primer pair EW01 is shown as SEQ ID NO.1 to SEQ ID NO.4; primer pair EW02 is shown as SEQ ID NO.5 to SEQ ID NO.8; primer pair EW03 is shown as SEQ ID NO.9 to SEQ ID NO.12; primer pair EW04 is shown as SEQ ID NO.13 to SEQ ID NO.16; primer pair EW05 is shown as SEQ ID NO.17 to SEQ ID NO.20; primer pair EW06 is shown as SEQ ID NO.21 to SEQ ID NO.24; primer pair EW07 is shown as SEQ ID NO.25 to SEQ ID NO.28; primer pair EW08 is shown as SEQ ID NO.29 to SEQ ID NO.32; primer pair EW09 is shown as SEQ ID NO.33 to SEQ ID NO.36; and primer pair EW10 is shown as SEQ ID NO.37 to SEQ ID NO.

40.

3. A universal nuclear gene molecular marker for earthworms, characterized in that: The method is formed by amplification using the molecular marker primers described in any one of claims 1 to 2.

4. A universal nuclear gene molecular marker database for earthworms, characterized in that: It includes the earthworm universal nuclear gene molecular marker as described in claim 3.

5. Use of the universal earthworm nuclear gene molecular marker primers according to claim 1 or 2, the universal earthworm nuclear gene molecular markers according to claim 3, and the universal earthworm nuclear gene molecular marker database according to claim 4 in the preparation of reagents for identifying earthworm germplasm resources and categories.

6. A method for identifying earthworm species, characterized in that: The steps include: Step S1, obtaining DNA of the earthworm to be identified; Step S2, performing PCR amplification using the primers described in claim 1; Step S3, performing high-throughput sequencing on the PCR amplification product obtained in step S2 and comparing it with the earthworm universal nuclear gene molecular marker database described in claim 4, obtaining the species information of the earthworm to be identified based on the molecular markers in the molecular marker database that match the earthworm to be identified and the phylogenetic tree established based on the molecular marker database.

7. A kit, characterized in that It comprises the earthworm universal nuclear gene molecular marker according to claim 3 and / or the earthworm universal nuclear gene molecular marker primer according to claim 1 or 2.

8. The kit according to claim 7, characterized in that It also includes reagents required for the PCR amplification system of the earthworm universal nuclear gene molecular marker primers according to claim 1 or 2.

9. A phylogenetic model, characterized in that The earthworm universal nuclear gene molecular marker database is constructed using the earthworm universal nuclear gene molecular marker primers according to claim 1 or 2, the earthworm universal nuclear gene molecular marker according to claim 3 and / or the earthworm universal nuclear gene molecular marker database according to claim 4.

10. The phylogenetic model according to claim 9, characterized in that Includes phylogenetic tree.

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