Method for constructing kelp core germplasm and identifying germplasm based on SNP (Single Nucleotide Polymorphism) molecular marker

Abstract

The invention provides a method for constructing a kelp core germplasm and identifying the germplasm based on an SNP (Single Nucleotide Polymorphism) molecular marker, which comprises the following steps: collecting kelp gametophyte germplasm with complete and accurate source information, identifying and screening the SNP marker, and determining an optimal core germplasm group by utilizing an EN-MR / CE model and comparing genetic diversity; 130 SNP marker sets which have polymorphism and are relatively uniformly distributed on chromosomes are further screened, so that 91 core germplasm can be effectively distinguished. The method can reflect genetic diversity of germplasm resources in a kelp germplasm bank, efficiently select core germplasm, accurately identify germplasm materials, and provide technical support for improving kelp germplasm preservation and utilization efficiency.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology, genomics and germplasm resources, and particularly relates to a method for constructing a core germplasm of Saccharina japonica and germplasm identification based on SNP molecular markers. Background Art

[0002] Saccharina japonica is a kind of large marine brown alga with important economic value. Alginate, mannitol and iodine produced from Saccharina japonica are widely used in many fields such as cosmetics, chemical industry, medicine and bioenergy. In addition, as an important part of the marine ecological environment, the kelp forest formed by Saccharina japonica can provide habitats, food and shelters for organisms, and has the functions of improving the biodiversity in the coastal waters and regulating the ecological environment of the coastal waters.

[0003] Saccharina japonica is the large alga with the longest breeding history and the highest yield in China.

[0004] The germplasm resources of Saccharina japonica are the core biological capital to support genetic breeding and the sustainable development of the industry. As a representative species of the large brown algae group, the preservation of Saccharina japonica germplasm is not only related to the potential of variety improvement, but also a strategic reserve for coping with climate change, disease outbreaks and marine environmental fluctuations. Saccharina japonica has an alternation of generations life history. The establishment of the gametophyte cloning technology of Saccharina japonica enables the long-term in-vivo preservation of Saccharina japonica gametophytes, so Saccharina japonica gametophytes have become the main objects of Saccharina japonica germplasm preservation. At present, scientific research institutions such as the Institute of Oceanology, Chinese Academy of Sciences, Ocean University of China, and Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, as well as high-tech enterprises such as Shandong Oriental Ocean Sci-Tech Co., Ltd. have all established Saccharina japonica gametophyte germplasm banks. However, there are many problems in the current Saccharina japonica germplasm bank: (1) Since this species has no natural distribution in China, the germplasm resources of Saccharina japonica in China are limited, the genetic diversity is low, and the wild genetic germplasm resources that can be exploited are limited. (2) A large number of Saccharina japonica gametophytes from the same variety (line) sources are preserved in the germplasm bank, which may cause redundancy of germplasm materials and result in a waste of a large amount of manpower and resources during the daily preservation process; (3) The source information of the preserved gametophyte germplasm materials is incomplete or inaccurate. For example, the gametophytes are not isolated by the personnel of the germplasm bank themselves, and the information of the germplasm materials obtained through gifts and exchanges is incomplete; due to the sharing of the seedling raising workshop or aquaculture area during the seedling raising and aquaculture stages, different Saccharina japonica varieties are confused, so the information of the gametophyte germplasm resources from this source is inaccurate; (4) There is a lack of an accurate and efficient germplasm identification method for Saccharina japonica germplasm materials. Summary of the Invention

[0005] Aiming at the problems of lack of genetic information of gametophyte germplasm and low efficiency of germplasm resource preservation and utilization in the current preservation of kelp germplasm resources, the purpose of the present invention is to provide a method for constructing a core germplasm of kelp and germplasm identification based on SNP molecular markers, laying a foundation for improving the preservation and utilization efficiency of the kelp germplasm bank and realizing the accurate identification of kelp germplasm.

[0006] The present invention first provides an SNP marker of kelp derived from the whole genome scale, and the SNP marker is located at the 51st position of any sequence among the sequences of SEQ ID NO: 1-130;

[0007] Another aspect of the present invention also provides a use of the SNP marker, which is a molecular marker for kelp population identification;

[0008] The present invention also provides a method for identifying a kelp population, which is to use the above SNP marker for identification;

[0009] As a specific record of an embodiment, the kelp population is a cultivated strain CKH obtained by cross-breeding different geographical types of kelp between China and Korea, wherein the SNP marker is located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 65, or at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 130.

[0010] Another specific record of an embodiment, the kelp population is a cultivated strain JLH obtained by cross-breeding kelp (S. japonica) and long kelp (S. longissima); the SNP marker is any one or more of the following SNP sites:

[0011] 1) The SNP site located at the 2nd position of the nucleic acid fragment with the sequence of SEQ ID NO: 2;

[0012] 2) The SNP site located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 6;

[0013] 3) The SNP site located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 7;

[0014] 4) The SNP site located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 19;

[0015] 5) The SNP site located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 29;

[0016] 6) The SNP site located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 31;

[0017] 7) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 66;

[0018] 8) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 67;

[0019] 9) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 75;

[0020] 10) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 76;

[0021] 11) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 77;

[0022] 12) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 81;

[0023] 13) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 92;

[0024] 14) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 94;

[0025] 15) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 102;

[0026] 16) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 103;

[0027] 17) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 104;

[0028] 18) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 107;

[0029] 19) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 108;

[0030] 20) The SNP locus at position 51 of the nucleic acid fragment with the sequence SEQ ID NO: 120.

[0031] The present invention also provides a method for constructing a core collection and germplasm identification of Saccharina japonica based on SNP molecular markers, comprising the following steps:

[0032] 1) Obtain samples of Laminaria japonica gametophyte germplasm resources with complete and accurate information sources, extract genomic DNA from all samples, conduct DNA sample testing, library construction, whole genome resequencing, quality control, and obtain high-quality sequencing data;

[0033] 2) using the resequencing data obtained in step 1), aligning to the reference genome to detect SNP sites, and detecting and filtering out high-quality SNP data sets;

[0034] 3) Using the high-quality SNP data set obtained in step 2) as genetic information data, Core Hunter3 software was used to construct core germplasm collections with different proportions;

[0035] 4) Analyze the genetic diversity parameters of the core germplasm collections with different proportions in step 3), combine the germplasm resource background information, and finally determine the core germplasm of Laminaria japonica;

[0036] 5) Using the core germplasm set obtained in step 4), extract all SNP sites of its samples, filter and select sites with high polymorphism, and construct a core SNP marker set containing 130 sites;

[0037] 6) Generate DNA fingerprints based on the core SNP marker set to achieve unique identification of germplasm.

[0038] The core germplasm screening method and SNP marker provided by the present invention can be applied to the scientific and efficient management of the Laminaria gametophyte germplasm bank; and can also be applied to application fields such as Laminaria genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :Distribution of 130 SNP loci on Laminaria chromosomes;

[0040] Figure 2 : A DNA fingerprint map constructed based on the genotype information of 91 samples at 130 SNP loci. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with embodiments and drawings.

[0042] Example 1: Screening of SNP loci in Laminaria japonica and construction of gametophyte core collection

[0043] (1) Representative gametophyte germplasm resource samples of kelp preserved in the kelp germplasm bank, including cultivated strains (JLH) cultivated by crossing Saccharina japonica and S. longissima; cultivated strains (CKH) cultivated by crossing S. japonica of different geographical types in China and South Korea; cultivated strains (CC) cultivated based on S. japonica of Chinese geographical type; wild kelp (WD) in foreign or Chinese sea areas; other species of the genus Saccharina (OS); unknown origin (UN).

[0044] (2) Take 0.1 g of kelp gametophyte samples, extract genomic DNA using a plant genomic DNA kit, prepare a paired-end library with an insert size of approximately 300 bp using the MGIEasy Universal DNA Library Preparation Kit, and then perform whole-genome resequencing on the DNBSEQ-T7 sequencing platform.

[0045] (3) Perform quality control on the obtained raw sequencing data to obtain high-quality sequencing data and align it to the kelp reference genome. Use Gatk4 software for variant detection. Use vcftools software (parameters: --min-alleles 2 --max-alleles 2 --min-meanDP 1 --max-missing 0.75) and the Variant Filtration tool (parameters: QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0) to filter SNPs and obtain a high-quality SNP dataset. Further filter out low-frequency loci (MAF < 0.05), and finally obtain 4,589,933 high-quality SNP loci, which will be used for the construction of core germplasm.

[0046] (4) Based on the genotype data obtained in step (3), use the EN-MR / CE comprehensive scheme of Core Hunter3 software to obtain core germplasm sets at different ratios: starting from a sample size of 10 and increasing in gradients of 10 sample sizes. Analyze the genetic diversity parameters of each set (Table 1), and combine the background information of germplasm resources to finally determine the kelp core germplasm.

[0047] The genetic diversity parameters mentioned above are: Modified Rogers distance (MR), Cavalli-Sforza and Edwards distance (CE), Shannon's allelic diversity (SH), Expected heterozygosity (HE), and Allele coverage (CV).

[0048] The criteria for determining the core collection are: high genetic distance index and diversity index, and the value of allele coverage is 1.

[0049] Table 1: Genetic parameters of core collection at different quantities

[0050]

[0051] When the sampling quantity is 80, the obtained Modified Rogers distance, Cavalli-Sforza and Edwards distance, Shannon's allelic diversity, and Expected heterozygosity all approach the maximum value, and the value of allele coverage is 100%. These data are better than those of core collections with other quantities. Further, based on the background information of kelp germplasm resources, 11 materials are supplemented, and finally a core collection consisting of 91 germplasms from 47 strains is constructed (Table 2), accounting for 40% of the initial number of germplasms, which can effectively improve the preservation efficiency of the kelp germplasm bank.

[0052] Table 2: Information of 91 core germplasm samples

[0053]

[0054]

[0055]

[0056] Using SNP loci, the neighbor-joining method is used to construct the phylogenetic trees of the initial germplasm resources and core germplasm resources. The results show that the germplasm materials with the same origin are basically clustered in one evolutionary group, and the core collection shows the same clustering result as the initial germplasm material collection.

[0057] Example 2: Screening of core SNPs of kelp and identification of core germplasm

[0058] (1)Extract the SNP loci of the core germplasm collection samples (91), and screen the core SNPs through filtering conditions. The screening conditions are as follows: remove heterozygous loci, remove locus genotype missing data, only retain loci located on chromosomes, remove loci with minor allele frequency (MAF) < 0.34, remove loci with polymorphism information content (PIC) ≤ 0.35, remove linkage disequilibrium loci, and retain no other SNP loci within 50 bp before and after polymorphic loci. Through the above screening steps, a total of 130 core SNP loci ( Figure 1 ) were obtained, located at the 51st position of any sequence in SEQ ID NO: 1-130; the specific SNP sequence information is shown in Table 3.

[0059] Table 3: SNP Locus Information

[0060]

[0061]

[0062]

[0063]

[0064] (2) Further use 130 SNPs to construct the DNA fingerprint map of the core germplasm collection ( Figure 2 ). Pairwise comparison of each locus of 91 samples was performed through R script, and a matrix diagram of differential loci between samples was constructed.

[0065] (3) The results of the matrix of differential loci between samples showed that there were only 2 differential loci between only one pair of samples, only 3 differential loci between only one pair of samples, and the number of differential loci between the remaining samples was 8 or more, which could completely distinguish all materials in the core germplasm collection, showing good identification ability for kelp germplasm materials. Taking the samples with the fewest differential loci between samples as an example:

[0066] Both F21 and M20 are from the same strain CKH - Strain 2 and have the most similar genetic backgrounds. The SNP locus located at the 51st position of SEQ ID NO: 65 and the SNP locus located at the 51st position of SEQ ID NO: 130 can both successfully distinguish the F21 and M20 samples effectively (Table 4).

[0067] Table 4: SNP Locus Information for Distinguishing Germplasm Materials F21 and M20

[0068]

[0069] Example 3: Strain Identification Based on Core SNPs of Kelp

[0070] (1) Select the germplasm resources from the core germplasm bank that are derived from JLH-Strain 1 and JLH-Strain 6. Both JLH-Strain 1 and JLH-Strain 6 are cultivated strains obtained through the hybridization of Saccharina japonica and Saccharina longissima followed by multiple generations of self-crossing. Among them, the kelp of JLH-Strain 1 has the traits of longer thallus length and high temperature tolerance, and the kelp of JLH-Strain 6 has the trait of wider thallus width.

[0071] (2) Among the 130 core SNP sets screened by the present invention, there are 20 differential SNP sites between the two strains. Therefore, these differential SNPs can be used to successfully and effectively distinguish the two strains (Table 5).

[0072] Table 5: SNP site information for distinguishing JLH-Strain 1 and JLH-Strain 6

[0073]

[0074]

[0075] In summary, the present invention develops and screens SNP markers from representative gametophyte germplasm sources in kelp germplasm, and the constructed core germplasm of kelp has high genetic diversity and allele coverage. The number of core germplasm accounts for 40% of the initial germplasm number, which can improve the preservation efficiency of the germplasm bank; the present invention screens 130 core SNP markers, which can achieve accurate identification of 91 core germplasm materials of kelp gametophytes derived from 47 kelp strains.

Claims

1. A genome-wide SNP marker of Laminaria japonica, characterized in that: The SNP marker is located at the 51st position of any sequence in SEQ ID NO: 1-130.

2. Use of the SNP marker described in claim 1 in identifying kelp populations.

3. A method for identifying kelp populations, characterized in that: The method described is to use any one or more of the SNP markers described in claim 1 for identification.

4. The method according to claim 3, characterized in that The kelp population is a cultivated variety CKH bred by hybridization of different geographical types of kelp from China and South Korea.

5. The method according to claim 4, characterized in that The SNP marker is located at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO: 65, or at the 51st position of the nucleic acid fragment with the sequence of SEQ ID NO:

130.

6. The method according to claim 4, characterized in that The kelp population is a cultivated variety JLH bred by hybridization of Laminaria japonica and Laminaria longissima.

7. The method according to claim 6, characterized in that The SNP markers include any one or more of the following SNP sites: 1) The SNP site at position 2 of the nucleic acid fragment with the sequence of SEQ ID NO: 2; 2) a SNP site located at position 51 of the nucleic acid fragment of SEQ ID NO: 6; 3) a SNP site located at position 51 of the nucleic acid fragment of SEQ ID NO: 7; 4) a SNP site located at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 19; 5) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 29; 6) the SNP site at position 51 of the nucleic acid fragment having the sequence of SEQ ID NO: 31; 7) the SNP site at position 51 of the nucleic acid fragment having the sequence of SEQ ID NO: 66; 8) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 67; 9) the SNP site at position 51 of the nucleic acid fragment having the sequence of SEQ ID NO: 75; 10) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 76; 11) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 77; 12) a SNP site located at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 81; 13) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 92; 14) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 94; 15) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 102; 16) the SNP site at position 51 of the nucleic acid fragment with the sequence of SEQ ID NO: 103; 17) a SNP site at position 51 of the nucleic acid fragment of SEQ ID NO: 104; 18) a SNP site at position 51 of the nucleic acid fragment having a sequence of SEQ ID NO: 107; 19) the SNP site at position 51 of the nucleic acid fragment with the sequence of SEQ ID NO: 108; 20) The SNP site located at position 51 of the nucleic acid fragment with the sequence of SEQ ID NO:

120.

8. A method for constructing core germplasm of kelp and identifying germplasm based on SNP molecular markers, characterized in that: The method comprises the following steps: 1) Obtain kelp gametophyte germplasm samples with complete and accurate information sources, extract genomic DNA from all samples, conduct DNA sample testing, library construction, whole genome resequencing, quality control, and obtain high-quality sequencing data; 2) Using the resequencing data obtained in step 1), align it to the reference genome to detect SNP sites, and detect and filter out high-quality SNP data sets; 3) Using the high-quality SNP dataset obtained in step 2) as genetic information data, use Core Hunter3 software to construct core germplasm collections with different proportions; 4) For the core germplasm collections with different proportions in step 3), analyze their genetic diversity parameters, combine the germplasm resource background information, and finally determine the core germplasm of Laminaria japonica; 5) Using the core germplasm set obtained in step 4), extract all SNP sites of its samples, filter and select sites with high polymorphism, and construct a core SNP marker set; 6) Generate DNA fingerprints based on the core SNP marker set to identify the uniqueness of germplasm.

9. The method according to claim 8, characterized in that The method and conditions for achieving unique identification in step 6) are: constructing genotypes between samples through DNA fingerprinting, and performing pairwise difference analysis on the loci of all samples through R language scripts to ensure that the number of difference loci between the two samples is ≥2.

10. The method according to claim 8, characterized in that The SNP marker set is the SNP marker set according to claim 1.

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