A molecular marker for kelp culture strain and its application

Abstract

The present invention provides a SNP marker combination of kelp, wherein the SNP site in the SNP marker combination is located at the 50th position of any sequence of SEQ ID NO: 1-247; the present invention also provides an application of the SNP marker combination in identifying kelp strains in another aspect. The SNP marker provided by the present invention is derived from a kelp genome-wide SNP data set, which is different from the previous molecular markers in a specific region of the genome, and is more accurate in identifying the source of the sample. The SNP marker provided by the present invention can also be applied to application fields such as kelp genetic breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of algae germplasm identification, and specifically relates to a molecular marker of a kelp culture strain and an application thereof. Background Art

[0002] Kelp (Saccharina japonica) is naturally distributed in the northwest Pacific Ocean. It is an important cultivated seaweed in the world because its ingredients are important industrial raw materials. China introduced kelp cultivation in the 1920s. At present, China's kelp cultivation area and output rank first in the world. Researchers have also cultivated more varieties according to industrial needs. China is not the country of origin of kelp. Although there are many varieties, the genetic background is narrow, the differences in traits between kelp are getting smaller and smaller, and due to the mixed cultivation process, it is difficult to accurately identify kelp varieties by observing the macroscopic morphology.

[0003] Molecular marker technology can be used to accurately detect differences between varieties or strains because it can detect extremely rich genomes. Molecular marker technology has been used to identify varieties of land plants and seaweed such as tea trees, ryegrass and Phalaenopsis orchids. SNP (Single Nucleotide Polymorphism) markers are the most widely distributed and abundant DNA molecular markers in the genome. Compared with other molecular markers, their genotyping polymorphism is higher and more in line with the needs of identifying Chinese kelp varieties with similar genetic backgrounds. With the advancement of sequencing technology, the use of GBS (Genotyping by Sequencing) technology to analyze SNP loci may identify kelp strains faster and cheaper. Summary of the invention

[0004] Aiming at the defect that kelp cultured strains are difficult to identify in the seedling stage, the present invention provides molecular markers of kelp cultured strains and applications thereof. The molecular markers include 247 strain-specific SNP markers, and these molecular markers can be used to identify kelp strains.

[0005] The present invention first provides a SNP marker combination of Laminaria japonica, wherein the SNP site in the SNP marker combination is located at the 50th position of any sequence of SEQ ID NO: 1-247;

[0006] In yet another aspect, the present invention provides an application of the SNP marker combination, which is application in identifying kelp strains.

[0007] The present invention also provides a method for identifying kelp strains, which uses the above-mentioned SNP marker combination as a molecular marker for identification;

[0008] The method described is to use the neighbor-joining method to construct a phylogenetic tree to identify the population.

[0009] The SNP marker combination provided by the present invention can also be used to prepare products for identification of kelp strains;

[0010] The product is a molecular identification kit or chip.

[0011] The SNP markers provided by the present invention are derived from the whole genome scale SNP data set of Laminaria japonica, which is different from the previous molecular markers in specific regions of the genome, and can more accurately identify the source of samples. The SNP markers provided by the present invention can also be applied to the application fields of Laminaria japonica genetic breeding, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : The distribution map of the SNP sites of the present invention on chromosomes;

[0013] Figure 2 : Phylogenetic tree of kelp strains constructed based on SNP marker combinations.

[0014] Figure 3 : Similarity correlation analysis diagram, where the horizontal axis is the similarity between samples calculated using all sites; the vertical axis is the similarity between samples calculated using core sites. Upper left corner: Fitting curve formula, correlation coefficient and p value. DETAILED DESCRIPTION

[0015] The present invention is described in detail below in conjunction with embodiments and drawings.

[0016] Example 1: Screening of SNP sites and strain identification of Laminaria japonica

[0017] The kelp strains B013, HG, ZK1 and ZK2 were collected. HG is a representative strain of southern kelp, which has been cultivated more in Shandong in recent years and is relatively large. B013, ZK1 and ZK2 are Rongcheng strains, which are more suitable for the characteristics of the sea area of ​​Rongcheng, the main production area in Shandong. The cultivation sites of these four strains (Table 1) are either far or close, which is conducive to testing the feasibility of this technology.

[0018] Table 1: Kelp strains

[0019]

[0020] Take 3-5 cm long leaves of the kelp strain and extract genomic DNA using a plant genomic DNA extraction kit;

[0021] The library was constructed according to the simplified genome sequencing technology Super-GBS method, and the Raw Reads were screened to obtain Clean Reads; the screening criteria were: (1) removing the adapter sequence; (2) removing the Reads with N bases ≥ 5; (3) using 4 bases as the window size for sliding windows, and removing the Clean Reads whose average base quality value was less than 20;

[0022] GATK software was used for SNP detection, and the standard of QD (ratio of mutation quality value (Quality) divided by coverage depth (Depth)) ≥ 2.0 was used for filtering;

[0023] vcftools software was used for further filtering, with the following criteria: (1) retaining reads support depth of no less than 4 loci; (2) deleting loci with a minimum allele frequency (MAF) less than 0.01; and (3) retaining loci that could be typed in 80% of the individuals.

[0024] Screening was carried out in sequence according to the principles of high marker quality, strong representativeness, high material discrimination and strong specificity: samples for fingerprint analysis were screened from the given VCF file, sites with a depth lower than 4 were changed to missing and only di-allelic SNP sites were retained; normalization within the group: the sample genotypes within each variety were compared, if the missing rate of a certain SNP site was ≤30.0% and the genotype consistency rate was ≥70.0%, or the missing data was used to represent the genotype of the variety at the SNP site; sites without polymorphism were eliminated; sites with a missing rate greater than 30.000000000000004% were eliminated; sites with a MAF less than 0.01 were eliminated; sites with a Hardy-Weinberg test p value lower than 1e were eliminated -5 The sites with polymorphism information content (PIC) less than 0.1 were removed; linkage filtering: the sites were delinked using plink software with the parameter --indep-pairwise 50 10 0.99.

[0025] Through the above steps, a total of 247 SNP sites were screened, which were located at the 50th position of any sequence of SEQ ID NO: 1-247; the specific SNP sequence information is shown in Table 1.

[0026] Table 1: Information table of SNP sites

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] 247 SNP sites were screened and the phylogenetic tree was constructed using the neighbor-joining method. Figure 2 As shown, the 40 genotype materials can be divided into 4 groups, and the genotype materials of different strains are clustered together; the phylogenetic tree results show that the genotype groups of different strains are obviously differentiated.

[0035] Based on the analysis of 247 core SNPs loci, H E The mean value is 0.22, and the range is 0.219-0.469; H O The mean is 0.26, ranging from 0.25 to 0.75; the mean PIC is 0.20, ranging from 0.195 to 0.359. The values ​​are higher than those calculated based on total SNPs.

[0036] Figure 3 The similarity between samples calculated using the SNPs of the present invention was shown, with a correlation coefficient of r=0.99, showing a highly significant linear correlation (P<0.01), indicating that the screened SNP site set is representative and can be effectively used for kelp strain identification.

[0037] In summary, the present invention developed and screened SNP markers from the germplasm of four kelp strains, and screened out 247 core SNP site combinations from the overall SNP markers, which can accurately distinguish the four cultured kelp strains. The genetic diversity obtained by analyzing the screened SNPs sites is higher than the analysis results of all sequenced SNP sites, indicating that the similarity of all sites and core sites is extremely significantly correlated.

Claims

1. A method for identifying kelp culture strains, characterized in that: The method uses a SNP marker group as a molecular marker and constructs a phylogenetic tree using the neighbor-joining method to identify kelp culture strains. The SNP sites in the SNP marker group are located at the 50th position of the sequence of SEQ ID NO: 1-247.

2. The method for identifying kelp culture strains according to claim 1, characterized in that: The kelp culture strains are kelp strains cultured in Guanwu, Lianjiang, Lijiang, Rongcheng, and Lidao, Rongcheng.

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