Combination of SNP molecular markers for the spiny traits of sea cucumber and its application

Abstract

The present invention belongs to the field of molecular biology technology, and relates to the fields of genomics, molecular biology, bioinformatics and whole genome selection breeding technology, and is specifically a combination of SNP molecular markers for the thorny traits of sea cucumbers and its application. The SNP molecular marker combination is at least one site recorded as follows, wherein the nucleotide sequences corresponding to the sites are respectively shown as SEQID NO.1‑16. The molecular marker combination of the present invention can be used for molecular marker-assisted breeding of sea cucumbers, shorten the breeding period, significantly improve breeding efficiency, reduce breeding costs, and is conducive to the sustainable and healthy development of the sea cucumber breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and relates to the technical fields of genomics, molecular biology, bioinformatics and whole genome selection breeding, and specifically is a SNP molecular marker combination for imitating the thorny traits of sea cucumbers and its application. Background Art

[0002] Imitation sea cucumber ( Apostichopus japonicus ) is also known as sea cucumber, belongs to the phylum Echinodermata, class Holothuroideaia, order Synallactida, genus Simulacrum Apostichopus ). Its medicinal and edible properties make sea cucumbers very popular in the consumer market. However, the rising demand has led to the overfishing of sea cucumbers and the crisis of natural resource depletion, and the sea cucumber aquaculture industry has emerged. The country is based on agriculture, and agriculture is based on seeds. Since the 1990s, the shortage of good varieties has gradually become an important factor restricting the development of the marine aquaculture industry. Breeders from all over the world have actively explored new ideas and methods for breeding, focusing on the mechanism analysis and genetic improvement of economic traits. Compared with the livestock industry, the genetic improvement of aquatic seed industry has emerged relatively late. Traditional breeding techniques represented by selective breeding and hybrid breeding have greatly promoted the development of genetic breeding in the aquatic industry, but the phenomenon of inbreeding depression after multiple generations of breeding has restricted the further development of genetic improvement. The emergence of DNA molecular markers has brought revolutionary impacts on the study of aquaculture genetics. The update and iteration of various life science technologies have provided an information platform and a basis for in-depth research on the genetic mechanism and breeding improvement of aquatic economic traits from the whole genome level.

[0003] As one of the species with the largest single output value in marine aquaculture, the economic traits of sea cucumbers, such as weight, body length, size and number of warty feet (thorns), directly determine their quality and market price. The first new sea cucumber variety "Shuiyuan No. 1" bred in my country through hybrid breeding technology has increased the number of warty feet of adult sea cucumbers by 40%, achieving good economic and social benefits. Based on whole-genome resequencing, Ni Ping and others found that the number of warty feet, as a complex quantitative trait, is jointly acted by multiple genes and has medium heritability, which also illustrates the feasibility of molecular breeding for warty feet. At present, the selection and genetic improvement of sea cucumber varieties are still mainly based on traditional methods, and whole genome selection breeding is still in its early stages. It is an irreversible trend in the selection and breeding of sea cucumber varieties to promote the construction of an efficient and precise breeding system that combines traditional sea cucumber breeding with modern molecular breeding. Summary of the invention

[0004] The purpose of the present invention is to provide a combination of SNP molecular markers for sea cucumber spiny traits obtained based on whole genome association and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A combination of SNP molecular markers for the thorny trait of sea cucumbers, wherein the combination of SNP molecular markers is at least one of the following sites, wherein the sites are Chr7:10991359, Chr4:10242747, Chr7:21800280, Chr22:8451096, Chr4:1745272, Chr3:28967057, Chr10:17874992, Chr11:9282749, Chr10:17911272, Chr17:12587179, Chr1:15454665, Chr17:18867543, Chr10:8375118, Chr1:15257313, Chr1:15610928, Chr10:1937829, and the corresponding nucleotide sequences are shown in SEQ ID NO.1-16 respectively.

[0007] For the specific sequence, see Table 2, which marks the base sequence of 200 bp before and after each SNP marker, and there is a base variation at the 100th bp.

[0008] An application of the sea cucumber spiny trait SNP molecular marker combination in sea cucumber spiny trait molecular marker assisted breeding.

[0009] A kit for breeding sea cucumbers with spiny traits, comprising at least one SNP molecular marker.

[0010] A method for detecting the genotype of sea cucumber using molecular biological technology, designing primers according to the nucleotide sequences on both sides of the molecular marker, performing genotyping on the corresponding SNP sites through primer amplification, and thus verifying the genotype of the sea cucumber individual to be tested.

[0011] The method is applied in molecular marker-assisted breeding of sea cucumber spiny traits.

[0012] Compared with the prior art, the present invention has the following advantages.

[0013] Compared with traditional phenotype-based selection breeding and hybrid breeding methods, the improved variety selection based on the SNP molecular marker combination of the sea cucumber spiny trait of the present invention has a higher trait purification degree; compared with QTL positioning, it covers the SNP site information of the whole genome and has higher genome detection accuracy and reliability.

[0014] The SNP molecular marker combination of the sea cucumber spiny trait screened by the present invention can be used for molecular marker-assisted breeding of sea cucumbers, and selection can be performed at an early stage, thereby accelerating the breeding process, improving breeding efficiency, and reducing breeding costs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Manhattan plot for genome-wide association analysis of the spiny traits of sea cucumber. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the content of the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0017] Example 1 Screening of SNP molecular markers associated with the spiny nature of sea cucumbers based on genome-wide association analysis

[0018] (1) Population selection and phenotypic statistics.

[0019] Specifically, four full-sib families were constructed using four pairs of S. imitatis parents (family I: 54 thorns in the father and 40 thorns in the mother; family II: 27 thorns in the father and 23 thorns in the mother; family III: 65 thorns in the father and 25 thorns in the mother; family IV: 20 thorns in the father and 56 thorns in the mother) and fed at the Weihai Rushan Sea Cucumber Seaweed Breeding Base. A total of 271 healthy S. imitatis with no body damage were randomly selected, numbered and cultured individually, and the number of papillae of each sea cucumber was recorded by manual counting.

[0020] R software was used to perform basic descriptive statistics on the collected papillopod data, including maximum value, minimum value, mean, standard deviation and coefficient of variation.

[0021] Table 1 Statistical analysis of the data of the tubercle of the sea cucumber

[0022]

[0023] (2) DNA extraction and resequencing: The body wall tissue of the sea cucumber was collected and ground into powder with liquid nitrogen. The genomic DNA was extracted according to the phenol imitation method experimental process. The DNA quality was tested by agarose gel electrophoresis and NanoDrop2000. The qualified DNA samples were randomly sheared into fragments of 350 bp in length. The library was constructed after end repair, poly A tail addition, sequencing adapter addition, purification, PCR amplification and other steps. The effective concentration of the library was accurately quantified by Qubit and qRT-PCR (effective concentration > 2 nM). After the library passed the inspection, high-throughput sequencing was performed on the Illumina Nova 6000 platform. The sequencing mode was PE150 and the sequencing depth was 10×.

[0024] (3) Genome alignment and quality control: The raw data obtained from the sequencing machine was filtered and quality checked using FastP (v 0.20.0) and FastQC (v 0.10.1) software. The filtered high-quality reads were aligned with the new version of the reference genome of Apostichopus japonicus (https: / / figshare.com / articles / dataset / The_genome_annotation_files_of_Apostichopus_japonicus / 22140020) using BWA (v0.7.13) software, and the results were tested for variant sites and quality control using SAMTools (v1.16.1) software. Among the SNPs obtained, not all SNPs are polymorphic. Some SNPs have only one allele or the allele frequency is very low. Minor Allele Frequency (MAF) is usually selected as an indicator to filter and screen SNPs, and SNP sites below 0.05 are eliminated. Hardy–Weinberg Equilibrium (HWE) refers to the ideal state where the frequencies of alleles remain stable over generations. The commonly used threshold for HWE is 10 -4 , remove the inconsistent SNPs. SNP call rate refers to the proportion of samples with a certain SNP successfully measured in all samples. Sites with SNP call rate lower than 95% were removed. The retained variant sites were annotated using snpEff (v 4.3) software.

[0025] (4) Genome-wide association analysis: A mixed linear model (MLM) was used to perform association analysis between the spiny trait phenotypic data and SNP molecular markers. In order to correct for population stratification, PC1 (38.20%, PC2 33.07%, PC3 22.137%) was used as a covariate, and the kinship matrix was used as a random effect. Finally, each SNP locus obtained an association value (p-value). The genome-wide significance threshold line -log 10 The p-value was set to an empirical value of 5, and the SNP loci with significant association were extracted using the software, that is, the 16 SNP loci related to the thorny traits of sea cucumbers were obtained as shown in Table 2 (-log 10 (p-value)>7.0), which can be used for molecular breeding of sea cucumber spiny traits. The Manhattan plot of the association data was drawn using the R function “CMplot” ( Figure 1 ).

[0026] Table 2 Information of 16 SNP markers significantly associated with the spiny traits of A. japonicus

[0027]

[0028] Note: SNP, site number; Chr, chromosome number; pos, physical position; Ref, genotype before mutation; Alt, genotype after mutation; maf, minor allele frequency; -log 10 (p-value), association threshold.

[0029] Example 2 Validation of SNP molecular markers for the spiny trait of A. japonicus in other populations

[0030] Based on the SNP molecular marker combination screened in Example 1, the individuals of the verification group were tested. The verification group used was individuals of the same age cultivated in the same breeding environment in Rushan, Weihai. 96 individuals with resequencing data were randomly selected to count the number of spines one by one. The results of the relevant comparative analysis are shown in Table 3, among which the number of spines of individuals of type (TG) at the Chr7:21800280 site was significantly more than that of type (TT), indicating that the dominant genotype of this site is (TG); the number of spines of individuals of type (AG) at the Chr22:8451096 site was significantly more than that of type (AA), indicating that the dominant genotype of this site is (AG); the number of spines of individuals of type (CT) at the Chr3:28967057 site was significantly more than that of type (CC), indicating that the dominant genotype of this site is (CT); the number of spines of individuals of type (TG) at the Chr10:17911272 site was significantly more than that of type (TT), indicating that the dominant gene at this site is (TG). type is (TG); the number of spines of individuals of (GG) type at Chr17:12587179 is significantly more than that of (GA), indicating that the dominant genotype of this site is (GG); the number of spines of individuals of (GA) type at Chr1:15454665 is significantly more than that of (GG), indicating that the dominant genotype of this site is (GA); the number of spines of individuals of (CA) type at Chr17:18867543 is significantly more than that of (CC), indicating that the dominant genotype of this site is (CA); the number of spines of individuals of (AT) type at Chr1:15610928 is significantly more than that of (AA), indicating that the dominant genotype of this site is (AT).

[0031] Table 3 Comparative analysis of different genotypes of spiny trait SNP loci in the validation population

[0032]

[0033] Note: Lowercase letters indicate a significance level of 0.05; uppercase letters indicate a significance level of 0.01.

[0034] As can be seen from the above, by detecting the genotype of the 16 SNP loci combination described in the present invention, the thorny trait of the sea cucumber can be genetically evaluated and the variety can be screened at the genome level, the breeding accuracy can be improved, and the sea cucumber individuals with the thorny phenotype can be selected in the early stage of cultivation, thereby controlling the breeding process and saving breeding costs. Although the present invention has been described in detail above with general instructions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements can be made to it on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A combination of SNP molecular markers for the trait of sea cucumber spinyness, characterized in that : The SNP molecular marker combination is the sequences shown in SEQID NO.3, SEQID NO.4, SEQID NO.6, SEQID NO.9, SEQID NO.10, SEQID NO.11, SEQID NO.12, and SEQID NO.15, wherein the 100th base in SEQID NO.3 mutates to T / G, the 100th base in SEQID NO.4 mutates to A / G, the 100th base in SEQID NO.6 mutates to C / T, the 100th base in SEQID NO.9 mutates to T / G, the 100th base in SEQID NO.10 mutates to G / G, the 100th base in SEQID NO.11 mutates to G / A, the 100th base in SEQID NO.12 mutates to C / A, and the 100th base in SEQID NO.15 mutates to A / T.

2. An application of the SNP molecular marker combination for the sea cucumber spiny trait according to claim 1 in sea cucumber spiny trait molecular marker-assisted breeding.

3. A kit for breeding sea cucumbers with spiny traits, characterized in that: Contains at least one SNP molecular marker according to claim 1.

4. A method for detecting the genotype of the spiny trait of sea cucumber using molecular biological technology, characterized in that: According to claim 1, primers are designed based on the nucleotide sequences on both sides of the SNP molecular marker, and genotyping of the corresponding SNP site is performed through primer amplification, thereby verifying the genotype of the sea cucumber individual to be tested.

5. Application of the method according to claim 4 in molecular marker-assisted breeding of sea cucumber spiny traits.

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