SNP (Single Nucleotide Polymorphism) molecular marker combination related to stichopus japonicus polysaccharide traits and application thereof

Abstract

The invention relates to an SNP molecular marker combination related to stichopus japonicus polysaccharide traits and application, belongs to the field of molecular biology, and provides an SNP molecular marker combination related to the stichopus japonicus polysaccharide content screened based on a whole genome association analysis technology, and a detection kit and a chip developed based on the SNP molecular markers. In a stichopus japonicus breeding procedure, individuals with more than one character dominant mutation site of the SNP molecular marker are screened as parents, so that the stichopus japonicus breeding process can be promoted; or offspring seeds with more than one character dominant mutation site of the SNP molecular marker are selected for breeding in the breeding process, so that the content of polysaccharide in the stichopus japonicus can be remarkably increased, and the economic value and the market competitiveness of the stichopus japonicus are improved.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and aquaculture, and in particular relates to a SNP molecular marker combination related to polysaccharide traits of sea cucumber (Apostichopus japonicus) and an application thereof. Background Art

[0002] Sea cucumber (Apostichopus japonicus) is an important marine invertebrate widely distributed in coastal areas of East Asia, such as China, Japan, and South Korea. Due to its high nutritional value and multiple bioactive components, such as polysaccharides, collagen, and saponins, sea cucumber is widely used in medicine, health products, and beauty cosmetics. These bioactive components have been shown to have significant functions in anti-oxidation, anti-tumor, immunomodulation, and anti-aging, so research on these components has attracted much attention.

[0003] The polysaccharide content in sea cucumbers is affected by many factors, including genetic background and environmental conditions. Traditional research methods mainly focus on the extraction and functional analysis of components, but there is limited understanding of the synthesis pathways and genetic regulation mechanisms of these components in sea cucumbers. Since the content of these components varies significantly between individual sea cucumbers and their genetic mechanisms are complex, it is of great significance to explore how to reveal and utilize these differences through genetic methods.

[0004] However, as a marine invertebrate, the genetic research of sea cucumber is still in its early stages. Existing research focuses on its basic biological characteristics and ecological behavior, while the understanding of the genetic control mechanism of its functional components is still limited. Summary of the invention

[0005] The present invention provides a combination of SNP molecular markers related to the polysaccharide traits of sea cucumbers and their applications in view of the above technical problems. The present invention provides a combination of SNP molecular markers related to the polysaccharide content in sea cucumbers screened out based on the whole genome association analysis (GWAS) technology and its application in sea cucumber breeding and bioactive component development. The combination of SNP molecular markers significantly related to the polysaccharide content of sea cucumbers is screened and verified. These SNP markers can be used as tools for genetic improvement and breeding to increase the content of target bioactive components in sea cucumbers. In addition, these markers can also be applied to the identification and evaluation of sea cucumber germplasm resources, thereby guiding efficient germplasm resource management and industrial development.

[0006] The present invention is achieved through the following technical solutions:

[0007] A SNP molecular marker combination related to sea cucumber polysaccharide traits, the loci and physical location information of the SNP markers in the SNP molecular marker combination are shown in Table 1.

[0008] The present invention also provides an application of the SNP molecular marker combination. The application method is to screen individuals with one or more SNP molecular marker dominant gene sites as parents in the sea cucumber breeding program, or to select seedlings with one or more SNP molecular marker dominant gene sites for breeding during the breeding process.

[0009] A kit for detecting polysaccharide traits in sea cucumbers, the kit comprising detection primers developed based on the SNP molecular marker.

[0010] A SNP chip for detecting polysaccharide traits in sea cucumbers, wherein the SNP chip is provided with probes for detecting the SNP molecular marker combination.

[0011] The beneficial effects of the present invention compared with the prior art are as follows:

[0012] The use of the SNP molecular marker combination of the present invention for the breeding of specific traits can significantly increase the polysaccharide content in sea cucumbers, and enhance the economic value and market competitiveness of sea cucumbers. At the same time, the SNP molecular marker combination of the present invention can also be widely used in the management and industrial development of sea cucumber germplasm resources, promoting the sustainable development of the sea cucumber industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the Manhattan plot and QQ plot of genome-wide association analysis of sea cucumber polysaccharide traits. DETAILED DESCRIPTION

[0014] The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

[0015] The present invention provides a combination of SNP molecular markers related to the polysaccharide content trait of sea cucumber screened based on genome-wide association analysis (GWAS) and an application method thereof.

[0016] Example 1 Screening of SNPs associated with sea cucumber polysaccharides based on genome-wide association analysis.

[0017] The following are the specific implementation steps:

[0018] (1) Sample collection and phenotypic statistics

[0019] Sample collection: 143 sea cucumbers were collected from seven areas including Bangchuidao, Changhai County, Huangcheng Island, Jimingdao, Jinzhou, and Lushun Wafangdian. The polysaccharide content of sea cucumbers was determined by the sulfuric acid method (optimization of the phenol-sulfuric acid method for rapid determination of polysaccharides).

[0020] (2) DNA extraction and resequencing

[0021] DNA was extracted from the inner body wall tissue of sea cucumbers using the TIANGEN amp Marine Animals DNA Kit (Beijing, China). Agarose gel electrophoresis was used to analyze the degree of DNA degradation and the presence of stray bands, RNA, and protein contamination. Qubit 2.0 was used to accurately quantify DNA concentration. DNA LibraryPrepKit for ILM kit was used to construct resequencing library for DNA that passed the quality inspection. After the library was constructed, Qubit2.0 was used for preliminary quantification, and the effective concentration of the library was accurately quantified using qPCR to ensure the quality of the library. After the library quality inspection was qualified, sequencing was performed using BGI MGI-2000 / MGI-T7 sequencing platform, and the sequencing mode was PE150 mode.

[0022] The original sequenced reads obtained by sequencing contain low-quality reads with adapters. To ensure the quality of information analysis, the raw reads were filtered to obtain clean reads, which were used for subsequent analysis. The raw reads were filtered using the software fastp (version 0.20.0).

[0023] Use the software BWA (mem alignment method) to align the clean reads after quality control with the reference genome sequence. The position of the clean reads on the reference genome can be located through alignment.

[0024] The NOVAR software tool is used to perform functional annotations on the detected gene variants. Based on the location of the variant site on the reference genome and the gene location information on the reference genome, the region where the variant site occurs in the genome (intergenic region, intron region or CDS region, etc.) and the impact of the variant (synonymous non-synonymous mutation, etc.) can be obtained. GATK is used to perform population SNPs detection on the samples, and the results are filtered to obtain reliable SNPs, and the population SNPs results are saved in VCF format.

[0025] (3) Genome-wide association study (GWAS)

[0026] The general linear model (GLM) and mixed linear model (LMM) were used for genome-wide association analysis, and the significance threshold was set at -log10(P)>6. 34 SNP loci associated with polysaccharide content were screened out, as shown in Table 1. The Manhattan plot and QQ plot of genome-wide association analysis of polysaccharide traits are shown in Figure 1 .

[0027] Table 1 SNP loci significantly associated with sea cucumber polysaccharide traits

[0028]

[0029]

[0030] Note: Ref is the conventional site, and mut is the dominant mutation site of the trait.

[0031] Table 2 200 bp base sequence before and after the SNP site

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] Example 2 Verification of SNPs Related to Sea Cucumber Polysaccharide Traits

[0042] The individuals of the validation group were tested based on the SNP molecular marker combination screened in Example 1. The validation group used was the same batch of samples collected, and 100 individuals with resequencing data were selected. The relevant comparative analysis results are shown in Table 3. Among the polysaccharide traits of sea cucumbers, the polysaccharide content of the (AA) type individuals at the Chr10:10138633 site was the highest, indicating that the dominant genotype of this site was (AA); the polysaccharide content of the (TT) type individuals at the Chr2:19339577 site was the highest, indicating that the dominant genotype of this site was (TT); the polysaccharide content of the (AC) type individuals at the Chr12:25010039 site was significantly higher than that of the (AA) type individuals, indicating that the dominant genotype of this site was (AC); the polysaccharide content of the (G) type individuals at the Chr12:25010053 site was significantly higher than that of the (G) type individuals, indicating that the dominant genotype of this site was (G) The polysaccharide content of T)) type individuals is significantly higher than that of (GG) type individuals, indicating that the dominant genotype of this site is (GT); the polysaccharide content of (AA) type individuals at Chr19:1757793 is the highest, indicating that the dominant genotype of this site is (AA); the polysaccharide content of (CC) type individuals at Chr15:15946459 is the highest, indicating that the dominant genotype of this site is (CC); the polysaccharide content of (AA) type individuals at Chr5:24071413 is the highest, indicating that the dominant genotype of this site is (AA); the polysaccharide content of (CC) type individuals at Chr15:5717747 is the highest, indicating that the dominant genotype of this site is (CC) ; The polysaccharide content of (TT) type individuals at the Chr21:18817455 site is the highest, indicating that the dominant genotype of this site is (TT); the polysaccharide content of (TT) type individuals at the Chr5:2691446 site is the highest, indicating that the dominant genotype of this site is (TT); the polysaccharide content of (GG) type individuals at the Chr19:1757835 site is the highest, indicating that the dominant genotype of this site is (GG); the polysaccharide content of (TT) type individuals at the Chr11:6790624 site is the highest, indicating that the dominant genotype of this site is (TT); the polysaccharide content of (GG) type individuals at the Chr15:3121764 site is significantly higher than that of (AA) type individuals The dominant genotype of this site is (GG); the polysaccharide content of (CT) type individuals at Chr20:6964976 is significantly higher than that of (CC) type individuals, indicating that the dominant genotype of this site is (CT); the polysaccharide content of (GG) type individuals at Chr5:24071395 is the highest, indicating that the dominant genotype of this site is (GG); the polysaccharide content of (CT) type individuals at Chr2:34206233 is significantly higher than that of (CC) type individuals, indicating that the dominant genotype of this site is (CT); the polysaccharide content of (AA) type individuals at Chr7:25902643 is the highest, indicating that the dominant genotype of this site is (AA);The polysaccharide content of (GA) type individuals at Chr15:3123651 was significantly higher than that of (GG) type individuals, indicating that the dominant genotype of this site is (GA); the polysaccharide content of (AA) type individuals at Chr12:23887980 was the highest, indicating that the dominant genotype of this site was (AA); the polysaccharide content of (AA) type individuals at Chr22:11695188 was the highest, indicating that the dominant genotype of this site was (AA); the polysaccharide content of (CT) type individuals at Chr7:3127318 was the highest, indicating that the dominant genotype of this site was (AA); The polysaccharide content of (TT) type individuals at Chr2:27350412 was significantly higher than that of (AA) type individuals, indicating that the dominant genotype of this site was (CT); the polysaccharide content of (TT) type individuals at Chr2:27350412 was the highest, indicating that the dominant genotype of this site was (TT); the polysaccharide content of (AC) type individuals at Chr11:28739868 was significantly higher than that of (AA) type individuals, indicating that the dominant genotype of this site was (AC); the polysaccharide content of (GT) type individuals at Chr15:23681825 was significantly higher The polysaccharide content of (GG) type individuals at Chr13:16768309 was the highest, indicating that the dominant genotype of this site was (GT); the polysaccharide content of (CC) type individuals at Chr2:41484812 was significantly higher than that of (CC) type individuals, indicating that the dominant genotype of this site was (CA); the polysaccharide content of (GA) type individuals at Chr2:41484825 was significantly higher than that of (GG) type individuals, indicating that the The dominant genotype of the site is (GA); the polysaccharide content of the (CC) type individuals at the Chr6:25384154 site is the highest, indicating that the dominant genotype of this site is (CC); the polysaccharide content of the (TG) type individuals at the Chr17:4072850 site is significantly higher than that of the (TT) type individuals, indicating that the dominant genotype of this site is (TG); the polysaccharide content of the (AG) type individuals at the Chr8:2296426 site is significantly higher than that of the (AA) type individuals, indicating that the dominant genotype of this site is (AG). ;

[0043] Table 3 Comparative analysis of SNP loci related to sea cucumber polysaccharide content in different genotypes in the validation population

[0044]

[0045]

[0046]

[0047]

Claims

1. A SNP molecular marker combination related to sea cucumber polysaccharide traits, characterized in that: The site and physical location information of the SNP markers in the SNP molecular marker combination are shown in the following table: Among them, Ref is the conventional site and mut is the trait dominant mutation site.

2. The use of the SNP molecular marker combination according to claim 1, characterized in that: The application method is to select individuals with one or more SNP molecular marker trait advantage mutation sites as parents in the sea cucumber breeding program, or to select seedlings with one or more SNP molecular marker trait advantage mutation sites for breeding during the breeding process.

3. A kit for detecting polysaccharide properties in sea cucumbers, characterized in that: The kit comprises detection primers developed based on the SNP molecular markers according to claim 1.

4. A SNP chip for detecting polysaccharide traits in sea cucumbers, characterized in that: The SNP chip is provided with probes for detecting the SNP molecular marker combination according to claim 1.

Images