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

Abstract

The invention relates to an SNP molecular marker combination related to stichopus japonicus collagen traits and application, belongs to the field of molecular biology, and provides an SNP molecular marker combination related to stichopus japonicus collagen content screened based on a whole genome association analysis technology, and a detection kit and a chip developed based on 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 collagen in the stichopus japonicus can be remarkably improved, 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 the collagen trait 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 collagen, collagenin, and saponin, 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 collagen 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 collagen trait of sea cucumbers and their application in view of the above technical problems. The present invention provides a combination of SNP molecular markers related to the collagen content in sea cucumbers screened out based on the whole genome association analysis (GWAS) technology and their application in sea cucumber breeding and bioactive component development. The combination of SNP molecular markers significantly related to the collagen 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 collagen traits, the sites 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 collagen traits in sea cucumbers, the kit comprising detection primers developed based on the SNP molecular marker.

[0010] A SNP chip for detecting collagen 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 selection of specific traits can significantly increase the content of collagen 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 collagen 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 trait of sea cucumber collagen content screened based on genome-wide association analysis (GWAS) and an application method thereof.

[0016] Example 1 Screening of SNPs associated with sea cucumber collagen 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 collagen content of sea cucumbers was determined using the double antibody sandwich method of the kit to determine the level of collagen (COL) in the samples.

[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] The clean reads after quality control were aligned with the reference genome sequence using the BWA software (mem alignment method). 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. 18 SNP loci associated with collagen content were screened out, as shown in Table 1. The Manhattan plot and QQ plot of genome-wide association analysis of collagen traits are shown in Figure 1 .

[0027] Table 1 SNP sites significantly associated with sea cucumber collagen traits

[0028]

[0029] Note: Ref is the conventional site, and mut is the dominant mutation site of the trait.

[0030] Table 2 200 bp base sequence before and after the SNP site

[0031]

[0032]

[0033]

[0034]

[0035] Example 2 Verification of SNPs Related to Sea Cucumber Collagen Traits

[0036] 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. In the sea cucumber collagen trait, the collagen content of the (TA) type individuals at the Chr14:837743 site was significantly higher than that of the (TT) type individuals, indicating that the dominant genotype of this site is (TA); the collagen content of the (CT) type individuals at the Chr23:5321767 site was the highest, indicating that the dominant genotype of this site is (CT); the collagen content of the (AT) type individuals at the Chr9:29847337 site was the highest, indicating that the dominant genotype of this site is (AT); Chr9 The collagen content of (AA) type individuals at Chr4:12134333 site is the highest, indicating that the dominant genotype of this site is (AA); the collagen content of (TT) type individuals at Chr4:11986718 site is the highest, indicating that the dominant genotype of this site is (TT); the collagen content of (GA) type individuals at Chr4:33221664 site is the highest, indicating that the dominant genotype of this site is (GA); the collagen content of (AG) type individuals at Chr3:12874796 site is the highest, indicating that the dominant genotype of this site is (AG); the collagen content of (CC) type individuals at Chr5:28920036 site is the highest. The collagen content is the highest, indicating that the dominant genotype of this site is (CC); the collagen content of the (CC) type individuals at the Chr7:11986718 site is the highest, indicating that the dominant genotype of this site is (CC); the collagen content of the (AA) type individuals at the Chr11:4077160 site is the highest, indicating that the dominant genotype of this site is (AA); the collagen content of the (AC) type individuals at the Chr1:41637966 site is the highest, indicating that the dominant genotype of this site is (AC); the collagen content of the (CC) type individuals at the Chr20:12181190 site is significantly higher than that of the (CT) type individuals. It indicates that the dominant genotype of this site is (CC); the collagen content of (CC) type individuals at Chr15:12587508 is the highest, indicating that the dominant genotype of this site is (CC); the collagen content of (TG) type individuals at Chr10:15601918 is the highest, indicating that the dominant genotype of this site is (TG); the collagen content of (TT) type individuals at Chr20:15012111 is the highest, indicating that the dominant genotype of this site is (TT); the collagen content of (GA) type individuals at Chr6:23347864 is the highest, indicating that the dominant genotype of this site is (GA).

[0037] Table 3 Comparative analysis of SNP loci related to the collagen content trait of sea cucumbers among different genotypes in the validation population

[0038]

[0039]

[0040]

Claims

1. A SNP molecular marker combination related to sea cucumber collagen 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 collagen 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 collagen properties in sea cucumbers, characterized in that: The SNP chip is provided with probes for detecting the SNP molecular marker combination according to claim 1.

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