Sipunculus nudus InDel molecular marker primer group and application thereof

Abstract

The invention relates to the technical field of molecular markers, in particular to a sipunculus nudus InDel molecular marker primer group and application thereof, 16 pairs of InDel primer pairs are developed based on a sipunculus nudus genome InDel marker, and the average allele of a sipunculus nudus population using the InDel marker is 8.875; the number of effective alleles is 4.110 on average; the average value of the polymorphic information content (PIC) is 0.692, the 16 primer pairs have the characteristic of high polymorphism, genetic analysis and clustering analysis can be effectively carried out on sipunculus nudus by applying the 16 primer pairs, and a solid foundation and a theoretical foundation are laid for improving the utilization of sipunculus nudus germplasm resources in the later period.

Description

[Technical field]

[0001] The invention relates to the technical field of molecular markers, and in particular to a Sipunculus quadrilateral InDel molecular marker primer set and an application thereof. [Background technology]

[0002] Siegeworm, also known as sandworm, belongs to the marine siegeworm family of the Siegeworm family. It is widely distributed in coastal areas of Guangdong, Fujian, Guangxi, Hainan and other places in my country. The genetic resources of sandworm are particularly abundant in the coastal areas of Guangxi. It generally lives in the intertidal mudflats or sandy seabeds along the coast. It emerges at high tide and lurks in sand and mud holes at low tide. It feeds on algae, organic matter and plankton. Modern medical research shows that siegeworm has a variety of effects, including anti-fatigue, promoting wound healing, enhancing cellular immunity, delaying aging, lowering blood pressure and antioxidant activity.

[0003] With the improvement of people's living standards and changes in dietary structure, the market demand for seafood such as sandworms has continued to increase. In order to meet market demand, the catch has also increased accordingly. Overfishing has led to the failure of natural resource protection and serious damage to the marine ecosystem. Therefore, it is of great significance to carry out genetic diversity research and natural resource protection of naked star worms.

[0004] Genetic diversity refers to the amount of genetic variation between individuals of a variety or a population within a species. It is the product of recombination, mutation, gene flow, and genetic drift of genetic material (DNA) during the inheritance process, which leads to changes in DNA sequence, epigenetic characteristics, protein structure or isozymes, physiological characteristics, and morphological characteristics. At present, there have been some reports on the study of genetic diversity of sandworms. There is a high level of genetic variation within the sandworm population, and there is no obvious genetic differentiation between populations. Low levels of genetic diversity between populations have also been reported. The main factor causing different conclusions may be the type of molecular marker. Because RAPD marker primers are short (10nt), have more binding sites, and lack stability. The mitochondrial genome of animals is conservative, and its markers are not ideal markers for discovering genetic diversity.

[0005] Insertions and deletions (InDels) are a common form of polymorphism that corresponds to the addition or removal of base pairs in the DNA sequence of an organism. InDels can also change the expression of genes by changing the phase and spacing of DNA sequences in the promoter region. These InDel markers are often associated with important traits.

[0006] The advent of genome-wide resequencing (WGRS) has paved the way for the generation of large amounts of sequence data at a relatively economical cost, thereby unlocking genetic markers that were hitherto inaccessible. Single nucleotide polymorphisms (SNPs) and InDels have become the most common types of genetic markers identified through WGRS. Medium-sized InDel markers can be efficiently used for PCR amplification and electrophoresis to detect polymorphisms. These markers have been used in genetic studies of a variety of animals, including goats and chickens. However, in sandworms, no InDels have been reported to date.

[0007] We will explore InDel markers of sandworms and discover the genetic diversity and genetic structure along the Beibu Gulf coast of China. [Summary of the invention]

[0008] In view of the above, it is necessary to develop InDel markers for the genome of Siegespiele and verify the molecular markers; using the developed InDel markers to conduct genetic diversity and clustering analysis of Siegespiele populations in different regions can play a huge role in the fields of molecular assisted genetic breeding, population genetic analysis, map-based cloning, gene localization and construction of genetic maps of Siegespiele.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A primer set for Sipunculus quadrilateral InDel molecular marker, comprising 16 pairs of InDel primers with nucleotide sequences as shown in SEQ ID NO.1-32.

[0011] The present invention also includes a kit comprising the Sipunculus quadripod InDel molecular marker primer set.

[0012] Furthermore, it also includes reagents required for PCR reaction and / or electrophoresis.

[0013] The present invention also includes the use of the Siegespinosa InDel molecular marker primer set or the kit in Siegespinosa cluster analysis and / or genetic diversity analysis.

[0014] The present invention also includes the use of the Siegespinosa InDel molecular marker primer set or the kit in distinguishing Siegespinosa germplasms from different regions.

[0015] Furthermore, the different regions are Fangchenggang City, Guangxi Zhuang Autonomous Region, Zhanjiang City, Guangdong Province, Qinzhou City, Guangxi Zhuang Autonomous Region, Hainan Province and Tieshan Port District, Beihai City, Guangxi Zhuang Autonomous Region. The present invention has the following beneficial effects:

[0016] 1. The present invention develops 16 pairs of InDel primer pairs based on the InDel markers of the Sipuncula genome. The average allele number of the Sipuncula population using the InDel markers is 8.875; the average number of effective alleles is 4.110; the average value of the polymorphic information content (PIC) is 0.692, which has the characteristics of high polymorphism. The application of the above 16 pairs of primer pairs can effectively perform genetic analysis and cluster analysis on Sipuncula, laying a solid foundation and theoretical foundation for improving the utilization of Sipuncula germplasm resources in the later stage.

Brief Description of the Drawings

[0017] Figure 1 The distribution of In Del markers in Sipuncula on 17 chromosomes of Sipuncula;

[0018] Figure 2 Cluster analysis of Siempuri spp. populations; green represents the Zhanjiang population, blue represents the Fangchenggang population, red represents the Hainan population, yellow represents the Tieshangang population, and purple represents the Qinzhou population;

[0019] Figure 3 The number of Sipunculus subpopulations (K); in the figure, the maximum value of K is 4, which divides the population into 4 subpopulations;

[0020] Figure 4 The population structure of the genetic resources of Sipunculus square. Subpopulation 1 includes 68 samples (ZJ1-ZJ50, FCG1-FCG15, HN1-HN3, red 1-68); subpopulation 2 includes 29 individuals (HN3-HN32, yellow 69-97); subpopulation 3 includes 39 individuals (TSG1-TSG39, green 98-136); subpopulation 4 includes 17 individuals (QZ1-QZ17, blue 137-153). [Specific implementation method]

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0022] Embodiment 1:

[0023] The molecular marker of Sipunculus square was constructed. The specific experimental method is as follows:

[0024] 1. Materials and Methods

[0025] 1. Identification of InDel markers: 18 transcriptomes from three tissues were sequenced and deposited in the genome repository of the National Genomics Data Center (accession number: GWHFDRQ00000000.1; https: / / ngdc.cncb.ac.cn / gwh). The quality of these sequences was assessed using FastQC software. Low-quality reads, such as reads containing ≥10% unknown nucleotides (N), reads with barcode adapters, and reads with more than 50% base quality scores below 20 were removed. High-quality reads were then aligned to the genome using Hisat2. Variant calling was performed using the default parameters of the Unified Genotyper module of the Genome Analysis Toolkit (GATK). InDels were extracted by the command line: gatkSelectVariants--select-type-to-including INDEL. According to the InDel identification results, InDel primers were designed using primer3. 85 pairs of primers with the same or similar melting temperatures (Tm) were randomly selected and synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0026] 2. Distribution of indels on chromosomes: To evaluate the distribution of indels on the genome, we counted the number of indels per 100 kb region on each chromosome. In addition, we also counted the number of indels based on the size variation of indels. To facilitate the description of the distribution of indels, we chose to keep only those indels located on chromosomes and remove the indels located on overlapping groups.

[0027] 3. Material collection and DNA extraction: The 153 individuals in this study included 50 samples from Zhanjiang City, Guangdong (ZJ1-ZJ50), 15 samples from Fangchenggang City, Guangxi (FCG1-FCG15), 32 samples from Danzhou City, Hainan (HN1-HN32), 39 samples from Tieshan Port area, Beihai (TSG1-TSG39), and 17 samples from Qinzhou City, Guangxi (QZ1-QZ17). All samples were collected and sent back to the laboratory. The genomic DNA of each sample was extracted using TIANamp Marine Animals DNA Kit (ID: DP324, Tiangen Biotechnology Beijing Co., Ltd.) according to the instructions, diluted to 10 ng / μl, and then stored at -20°C.

[0028] 4. InDel-PCR analysis: InDel-labeled polymerase chain reaction (PCR) was performed in a volume of 10 μL. The reaction system included 5 μL 2×TSINGKE Master Mix, 0.2 μL forward and reverse InDel primers (10 μmol / L), 10 ng DNA, and 3.6 μL ddH2O.

[0029] The PCR program was: 95°C for 4 min; 95°C for 45 s, Tm (primer dependent) for 45 s, 72°C for 45 s, 30 cycles; final extension at 72°C for 7 min, and storage at 4°C. PCR products were separated in polyacrylamide gels and visualized with silver staining. Clear bands were recorded manually for subsequent analysis.

[0030] 5. Genetic diversity and genetic structure: PowerMarkerV3.2.5 and GenAlEx6.5 software were used to analyze genetic diversity parameters such as the number of alleles (Na), the effective number of alleles (Ne), the polymorphic information content (PIC), the observed heterozygosity (Ho), the expected heterozygosity (He) and the Shannon index (I). The polygenic dendrogram was drawn using MEGA software. Principal component analysis (PCA) and estimation of genetic differentiation (Fst) between populations were performed using GenAlEx6.5. The genetic structure of the population was analyzed using a mixed model using Structure 2.3.4 software. The burn-in period was set to 10,000 and 100,000 MCMC replicates. For each K value, the analysis was run 15 times. The number of subpopulations (K) was assigned a range of 2 to 10. The most likely K value was determined by identifying the highest value of ΔK.

[0031] 2. The results obtained by the above method are as follows:

[0032] 1. The characteristics of genome-wide Indels are shown in Tables 1 and Figure 1 shown.

[0033] Table 1 Distribution of nDel size changes along 17 chromosomes

[0034]

[0035]

[0036] As shown in Table 1, a total of 168,771 InDels were identified in the genome. A total of 167,764 Indels were detected on 17 chromosomes. The distribution of InDels showed that they were distributed throughout the genome (Table 1 and Figure 1) are not uniform and scattered. Among these chromosomes, Chr11 has the most InDel sites, with a total of 14,724. Next is Chr03, with 13,732 sites; Chr01, with 13,636 sites; Chr04, with 12,628 sites; Chr02, with 12,441 sites; Chr06, with 10,854 sites; Chr12, with 10,081 sites; Chr13, with 9,501 sites; Chr15, with 8,991 sites; Chr09, with 8,869 sites; Chr07, with 8,351 sites; Chr16, with 8,078 sites; Chr14, with 7,781 sites; Chr10, with 6,595 sites; and Chr05, with 6,574 sites. In contrast, Chr17 has the least, with only 6,354 sites. Among all chromosomes, Chr01 had the highest density of InDels (141.187 / Mb), followed by Chr03 (138.540 / Mb), Chr04 (134.393 / Mb), Chr09 (134.120 / Mb), Chr02 (126.186 / Mb), Chr11 (125.226 / Mb), Chr06 (124.343 / Mb), Chr13 (115.531 / Mb), Chr15 (111.681 / Mb), and Chr12 (110. The density of indels was 116.639 / Mb.

[0037] 2. Screening of polymorphic InDels:

[0038] The applicant successfully designed 25,558 primers in total. Five primers were selected that were evenly distributed on each chromosome. A total of 85 primers were selected for synthesis and then screened in 6 DNA templates. The results showed that none of the three primers amplified any bands. In contrast, 82 primers amplified bands and one primer amplified a single band. The amplification success rate was 96.47% and the polymorphism ratio was 98.78%.

[0039] 3. Genetic diversity: The genetic diversity of 153 sandworm samples was evaluated using 16 highly polymorphic primers. The results are shown in Tables 2 and 3.

[0040] Table 2 16 InDel markers used in this study

[0041]

[0042] Table 3 Genetic diversity of sandworms using 16 InDel markers

[0043]

[0044] The results are shown in Table 3. A total of 142 alleles were detected. The number of alleles (Na) ranged from 4 to 15, with an average of 8.875. The effective number of alleles (Ne) ranged from 2.110 to 6.009, with an average of 4.110. The observed heterozygosity (Ho) ranged from 0.009 to 0.768, with an average of 0.232. The expected heterozygosity (He) ranged from 0.603 to 0.834, with an average of 0.734. The Shannon index (I) ranged from 0.919 to 2.085, with an average of 1.576. The polymorphism information content (PIC) ranged from 0.440 to 0.816, with an average of 0.692. All these results indicate that the genetic diversity of the population in this study is high.

[0045] Cluster analysis was performed using the above primers, and the results were as follows Figure 2 As shown in Figure 2, the clustering results show that the five groups of samples can be roughly divided into two categories ( Figure 2 ). The samples from HN (Hainan), QZ (Qinzhou) and TSG (Tieshangang) clustered into one group (the first group), indicating that the above provenances were closely related. At the same time, individuals from ZJ (Zhanjiang) and FCG (Fangchenggang) clustered into another group (the second group), indicating that the genetic relationship between these two provenances was the closest. It is worth noting that individuals from HN (Hainan) were divided into the second group.

[0046] Genetic structure Figure 3-Figure 4 As shown, this application uses Structure 2.3.4 software for genetic structure analysis. The optimal number of subpopulations is determined according to the DeltaK method. The results show that the K with the largest ΔK at the turning point is 4 ( Figure 3 ). It can be seen that this study divides the entire population into 4 subpopulations ( Figure 4 ). Subgroup 1 includes 68 samples (ZJ1-ZJ50, FCG1-FCG15, HN1-HN3, red); Subgroup 2 includes 29 individuals (HN1-HN29, yellow); Subgroup 3 includes 39 individuals (TSG1-TSG39, green); Subgroup 4 consists of 17 individuals (QZ1-QZ17, blue).

[0047] Genome-wide InDels markers: We found that InDel markers are distributed throughout the genome. This phenomenon was observed in both tobacco and rice, indicating that the results of these studies are consistent. Genome-wide InDels can provide a useful tool for genetic diversity and molecular marker-assisted breeding. Compared with lentils, InDels are unevenly distributed, with higher InDels density in some areas and lower InDels density in some areas. The reason may be two factors. On the one hand, the transcriptome samples used for InDel development are limited, which greatly limits the detection range. On the other hand, the transcriptome is the product of spatiotemporal gene expression and does not cover the entire genome. Finally, it may be related to the genome of sandworms and requires further study.

[0048] Availability of InDel markers: 82 of the 85 primer pairs screened were able to amplify products. Of these 82 primer pairs, 81 were polymorphic, indicating that this type of marker is suitable for genetic diversity evaluation of sandworms and molecular marker-assisted breeding. InDel markers have been reported in some model organisms such as goats and chickens, and according to previous reports, InDel markers are associated with important traits. This shows that InDel markers are very valuable.

[0049] Genetic diversity: Genetic diversity is the most important attribute of a population and is the basis for adaptation to the environment and genetic improvement. The two key elements for discovering genetic diversity are molecular markers and population sampling.

[0050] Although various molecular markers such as RAPDs and SSRs have been used in molecular studies of P. nakedus, the types of molecular markers are still relatively small compared with molecular markers of other species, which poses a challenge to the research of genetic resources.

[0051] This study used 16 polymorphic InDel markers to reveal the genetic diversity of sandworms. The average number of alleles in the population was 8.875, which is higher than the previously recorded 1.897. Therefore, we speculate that the InDel markers developed in this study are feasible for analyzing the genetic diversity of naked siphonophores.

[0052] Among the 16 InDel primers, six primers, Chr03-57264917, Chr04-71081678, Chr07-17389605, Chr08-9362134, Chr10-2788932, and Chr16-5274098, were found to have higher genetic diversity compared with other sites. This is because the generation of InDel sites usually involves mechanisms such as slippage mismatch and non-homologous recombination during DNA replication and transposable elements. These variations occur in some non-conserved genomic regions with a relatively high frequency, resulting in a large number of variations. In contrast, variations are selected in conservative genomic regions, resulting in relatively low genetic diversity. In general, InDels have no significant effect on the structure and function of proteins and are therefore subject to weak selection pressure. However, some InDels located in key functional regions of encoded proteins are subject to strong selection pressure. These variations may be associated with important traits.

[0053] Genetic structure: Differences in the genetic structure of populations reflect genetic diversity and the potential for a species to adapt to a changing environment. Genetic structure analysis based on allele frequencies can reveal information about the origin and composition of lineages and the exchange of genetic material.

[0054] This study divided the entire population into four subpopulations and detected the existence of genetic structure within the population.

[0055] As for the mating system, the sperm and eggs of sandworms are released into the seawater in a fluctuating state, which is mixed evenly, so the sperm and eggs may be considered to mate randomly. Therefore, the mating system may not be the main reason for the genetic structure.

[0056] Subpopulation 1 consists of three provenances, indicating the presence of gene flow. Gene flow generally includes two modes: the migration of germplasm resources and the migration of germ cells. Gametes move from one population to another with ocean currents. In other words, genetic resources are carried from one place to another by humans. This phenomenon was found in the cluster analysis. All of the above lead to changes in gene frequencies in the gene pool. Overfishing can also lead to changes in gene frequencies. Therefore, we speculate that human activities are the main cause of genetic structure.

[0057] In summary, this study identified a total of 168,771 InDels in the genome and designed 25,558 pairs of primers. A subset was synthesized and verified here. About 97.47% of the primers could be successfully amplified in sandworms. 16 InDels with high polymorphism revealed a high genetic diversity of the population, and also detected genetic differentiation and gene flow between provenances. Finally, the genetic structure was found and the population was divided into 4 subgroups. The results of this study will provide useful molecular markers and scientific basis for the efficient utilization and effective protection of the genetic resources of naked siphonophores.

[0058] In summary, the present invention utilizes the self-constructed Siegespinosa InDel markers with good polymorphism and high specificity to effectively perform genetic analysis and cluster analysis on Siegespinosa, which can lay a solid foundation and theoretical basis for improving the utilization of Siegespinosa germplasm resources in the future.

[0059] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A primer set for the InDel molecular marker of Sipunculus quadripunctatus, characterized in that: The primer set includes 16 pairs of InDel primers with nucleotide sequences as shown in SEQ ID NO.1-32.

2. A kit comprising the Sipunculus quadripod InDel molecular marker primer set according to claim 1.

3. The kit according to claim 2, characterized in that It also includes reagents required for performing PCR reactions and / or electrophoresis.

4. Use of the Sipunculus InDel molecular marker primer set according to claim 1 or the kit according to claim 2 or 3 in cluster analysis and / or genetic diversity analysis of Sipunculus.

5. Use of the InDel molecular marker primer set for Sipuncula as claimed in claim 1 or the kit as claimed in claim 2 or 3 in distinguishing Sipuncula germplasm from different regions.

6. The use according to claim 5, characterized in that The different regions mentioned are Fangchenggang City, Guangxi Zhuang Autonomous Region, Zhanjiang City, Guangdong Province, Qinzhou City, Guangxi Zhuang Autonomous Region, Hainan Province and Tieshan Port Area, Beihai City, Guangxi Zhuang Autonomous Region.

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