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. According to the method, kelp gametophyte germplasm with complete and accurate source information is collected, SNP site detection and screening are performed, and an optimal core germplasm group is determined by utilizing an EN-MR / CE model and combining genetic diversity comparison; 130 SNP markers which are uniformly distributed on chromosomes and rich in polymorphism are obtained through further screening, and accurate identification of 91 core germplasm can be realized. Meanwhile, the core SNP marker can be used for effectively distinguishing germplasm materials from different kelp cultivated strains. According to the method, the genetic diversity background of the kelp germplasm bank can be truly reflected, the core germplasm is efficiently screened, the germplasm material is identified, and technical support is provided for improving the storage and utilization efficiency of the kelp germplasm.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology, genomics, and germplasm resources, specifically to a method for constructing and identifying kelp core germplasm based on SNP molecular markers. Background Technology

[0002] Kelp (Saccharina japonica) is a large marine brown algae with significant economic and ecological value. Algin, mannitol, and iodine, produced from kelp, are widely used in cosmetics, chemicals, pharmaceuticals, and bioenergy. Furthermore, as an important component of the marine ecosystem, kelp forests provide habitats, food, and shelter for organisms, playing a role in enhancing nearshore biodiversity and regulating the nearshore marine environment.

[0003] Kelp is the oldest and most productive large algae species cultivated in my country. After being accidentally introduced to Dalian, my country from Hokkaido, Japan in the 1920s, wild populations were discovered there in the late 1930s. In the 1940s, Japanese algae expert Yoshiro Otuki and other Japanese farmers gradually introduced kelp to Dalian. In 1949, Yantai, Shandong Province, pioneered large-scale kelp cultivation using floating raft technology and artificial seedling raising techniques, which were gradually promoted to Qingdao. With the implementation of the "southern migration cultivation" strategy, kelp cultivation technology was introduced from Qingdao to Zhoushan, Zhejiang Province in 1956, and further promoted to Lianjiang, Fujian Province in 1957, propelling my country to become a core area for global kelp cultivation and production. After decades of kelp breeding research, my country has successively cultivated more than 40 new kelp varieties, 12 of which have been approved by the National Aquatic Breeding Committee, providing important support for the sustainable development of the kelp industry.

[0004] Germplasm resources are the core biological capital supporting genetic breeding and sustainable industrial development. As a representative species of large brown algae, the preservation of kelp germplasm resources is not only related to the potential for variety improvement, but also a strategic reserve for coping with climate change, disease outbreaks and marine environmental fluctuations. The life history of kelp has typical heteromorphic alternation of generations. The establishment of kelp gametophyte cloning technology has enabled the long-term live preservation of kelp gametophyte materials, making kelp gametophytes the main carrier for current kelp germplasm preservation. At present, research institutions such as the Institute of Oceanology of the Chinese Academy of Sciences, Ocean University of China, and Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences, as well as high-tech enterprises such as Shandong Oriental Ocean Technology Co., Ltd., have established kelp gametophyte germplasm banks, laying the foundation for germplasm resource preservation and utilization. However, there are still many problems in the management and utilization of existing kelp germplasm banks in my country: (1) Kelp is not naturally distributed in my country, so my country's kelp germplasm resources are limited, the level of genetic diversity is low, and the amount of wild genetic germplasm resources that can be tapped is limited. (2) The germplasm bank preserves a large number of kelp gametophytes from the same variety (line), which can easily lead to redundancy of germplasm materials and increase the consumption of manpower, material resources and space resources for daily preservation and management; (3) The source information of some preserved gametophyte germplasm materials is incomplete or inaccurate. For example, some gametophytes were obtained through donations, exchanges and other means, and the background information is missing; during the seedling and aquaculture stages, different varieties are mixed due to the sharing of seedling workshops or aquaculture areas, which further causes the subsequent separation and preservation of gametophyte germplasm information to be distorted; (4) In the existing technical system, there is a lack of efficient kelp germplasm identification methods, which makes it difficult to meet the needs of accurate identification of germplasm resources. Summary of the Invention

[0005] To address the current problems of insufficient genetic information in gametophyte germplasm and low efficiency in germplasm resource preservation and utilization, this invention aims to provide a method for constructing core kelp germplasm and identifying germplasm based on SNP molecular markers. This lays the foundation for improving the preservation and utilization efficiency of kelp germplasm banks and achieving accurate identification of kelp germplasm.

[0006] This invention first provides a genome-wide SNP marker for kelp, wherein the SNP marker is located at position 50 of any sequence in SEQ ID NO: 1-130;

[0007] In another aspect, the present invention also provides an application of the aforementioned SNP marker as a molecular marker for distinguishing kelp core germplasm samples;

[0008] The present invention also provides a method for identifying kelp cultivation varieties, wherein the above-mentioned SNP markers are used for identification;

[0009] As a specific example, the kelp core germplasm sample is a set of 91 core samples constructed based on genetic diversity and germplasm resource background information, and the SNP is marked as the 50th position of the sequence corresponding to SEQ ID NO: 1-130.

[0010] In another specific embodiment, the kelp population is a cultivated strain JLH, bred from a cross between kelp (S. japonica) and longissima; wherein the SNP marker is any one or more of the following SNP loci:

[0011] 1) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:2;

[0012] 2) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:6;

[0013] 3) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:7;

[0014] 4) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:19;

[0015] 5) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:81;

[0016] 6) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:92;

[0017] 7) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:94;

[0018] 8) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:102;

[0019] 9) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:103;

[0020] 10) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:104;

[0021] 11) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:107;

[0022] 12) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:108;

[0023] This invention also provides a method for constructing and identifying kelp core germplasm based on SNP molecular markers, comprising the following steps:

[0024] 1) Obtain kelp gametophyte germplasm resource samples with complete and accurate information sources, extract genomic DNA from all samples, and perform DNA sample testing, library construction, whole genome resequencing, and quality control to obtain high-quality sequencing data;

[0025] 2) Using the resequencing data obtained in step 1), SNP sites are detected by aligning to the reference genome, and high-quality SNP datasets are detected and filtered out.

[0026] 3) Using the high-quality SNP dataset obtained in step 2) as genetic information data, construct core germplasm sets with different proportions using Core Hunter3 software;

[0027] 4) For the core germplasm sets with different proportions in step 3), analyze their genetic diversity parameters and combine them with germplasm resource background information to finally determine the core germplasm of kelp;

[0028] 5) Using the core germplasm set obtained in step 4), extract all SNP loci from its samples, filter and select loci with high polymorphism, and construct a core SNP marker set containing 130 loci;

[0029] The core germplasm screening method and SNP markers provided by this invention can be applied to the scientific and efficient management of kelp gametophyte germplasm banks; they can also be applied to kelp genetic breeding and other fields. Attached Figure Description

[0030] Figure 1 Distribution map of 130 SNP loci on kelp chromosomes;

[0031] Figure 2 DNA fingerprinting based on genotypic information of 91 samples at 130 SNP loci. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0033] Example 1: Screening of SNP sites in kelp and construction of core germplasm of gametophytes

[0034] (1) Representative kelp gametophyte germplasm resources from the kelp germplasm bank were used as experimental materials. The sources of samples isolated from sporophytes included: cultivated strains bred from crosses of kelp (S. japonica) and longissima (JLH); cultivated strains bred from crosses of different geographical types of kelp (S. japonica) from China and South Korea (CKH); cultivated strains based on the geographical type of kelp (S. japonica) in China (CC); wild kelp from foreign or Chinese sea areas (WD); other species of the genus Kelp (OS); and unknown sources (UN).

[0035] (2) Take 0.1g of kelp gametophyte sample, extract genomic DNA using the plant genomic DNA kit, prepare a paired-end library with an insert size of about 300 bp using the MGIEAsy universal DNA library preparation kit, and perform whole-genome resequencing on the DNBSEQ-T7 sequencing platform.

[0036] (3) Quality control was performed on the raw sequencing data to obtain high-quality sequencing data, which were then aligned to the kelp reference genome. Variation detection was performed using Gatk4 software, and SNPs were filtered using vcftools software (parameters: --min-alleles 2 --max-alleles 2 --min-meanDP 1 --max-missing 0.75) and Variant Filtration tool (parameters: QD<2.0||FS>60.0||MQ<40.0||MQRankSum<-12.5||ReadPosRankSum<-8.0). Low-frequency sites with a minimum allele frequency (MAF) < 0.05 were further removed, resulting in 4,589,933 high-quality SNP sites for subsequent core germplasm construction.

[0037] (4) Based on the genotype data obtained in step (3), core germplasm sets with different sampling ratios were constructed using the EN-MR / CE integrated scheme of Core Hunter3 software: starting with a sample size of 10, the sets were constructed sequentially by adding 10 samples at each gradient. The genetic diversity parameters of each set were analyzed (Table 1), and combined with the background information of germplasm resources, the core germplasm of kelp was finally determined.

[0038] The genetic diversity parameters mentioned are: Modified Rogers distance (MR), Cavalli-Sforza and Edwards distance (CE), Shannon's allelic diversity (SH), expected heterozygosity (HE), and allele coverage (CV).

[0039] The criteria for determining core germplasm are: high genetic distance index and diversity index, and an allele coverage value of 1.

[0040] Table 1: Genetic parameters of core germplasm sets with different numbers of units

[0041] quantity Rogers genetic distance Cavalli-Edward genetic distance Shannon Genetic Diversity Index Expected heterozygosity Allele coverage 10 0.4260 0.4289 15.7302 0.2551 0.8958 20 0.4305 0.4334 15.7769 0.2808 0.9797 30 0.3734 0.3767 15.7756 0.2782 0.9899 40 0.3906 0.3939 15.7801 0.2811 0.9970 50 0.3691 0.3724 15.7613 0.2667 0.9980 60 0.3769 0.3803 15.7825 0.2822 0.9996 70 0.3786 0.3819 15.7796 0.2797 0.9999 80 0.3621 0.3658 15.7861 0.2843 1 90 0.3421 0.3458 15.7806 0.2800 1 100 0.3385 0.3422 15.7809 0.2802 1 110 0.3384 0.3421 15.7739 0.2747 1 120 0.3360 0.3398 15.7910 0.2878 1 130 0.3272 0.3310 15.7838 0.2822 1 140 0.3195 0.3233 15.7847 0.2827 1 150 0.3077 0.3118 15.7806 0.2800 1 160 0.3131 0.3170 15.7758 0.2761 1 170 0.3068 0.3108 15.7774 0.2773 1 180 0.3122 0.3162 15.7821 0.2808 1 190 0.3058 0.3098 15.7849 0.2829 1 200 0.2988 0.3029 15.7778 0.2775 1 210 0.2978 0.3019 15.7780 0.2777 1 220 0.2949 0.2991 15.7783 0.2778 1 226 0.2936 0.2977 15.7805 0.2795 1

[0042] When the sample size was 80, Rogers genetic distance, Cavalli-Edwards genetic distance, Shannon genetic diversity index, and expected heterozygosity all approached their maximum values, and the allele coverage was 100%, demonstrating superior overall performance compared to other core germplasm sets with fewer than 80 samples. Based on this, 11 additional materials were added using background information on kelp germplasm resources, ultimately constructing a core germplasm set containing 91 accessions (Table 2), approximately 40% of the initial germplasm count, which effectively improves the preservation efficiency of the kelp germplasm bank.

[0043] Table 2: Information on 91 core germplasm samples

[0044]

[0045]

[0046]

[0047] Using SNP loci, a phylogenetic tree of the initial germplasm resources and the core germplasm resources was constructed using the neighbor-joining method. The results showed that germplasm materials from the same origin basically clustered into one evolutionary group, and the core germplasm set showed the same clustering results as the initial germplasm material set.

[0048] Example 2: Screening of core SNPs in kelp and differentiation among core germplasm samples (91 samples)

[0049] (1) SNP loci were extracted from the core germplasm collection sample (91 loci), and core SNPs were screened using filtering conditions. The screening conditions were: removing heterozygous loci, removing loci with missing genotypes, retaining only loci located on chromosomes, removing loci with an allele frequency (MAF) < 0.34, removing loci with a polymorphism information content (PIC) ≤ 0.35, removing linkage disequilibrium loci, and retaining loci with no other SNP loci within 50 bp before and after them. Through the above screening steps, a total of 130 core SNP loci were obtained ( Figure 1 (), located at the 50th position of any sequence in SEQ ID NO: 1-130; specific SNP sequence information is shown in Table 3.

[0050] Table 3: SNP Locus Information Table

[0051] SNP locus number Chromosome numbering SNP base sequence (reference / variation) SNP site location 1 chr1 G / A 12297817 2 chr1 A / G 16095582 3 chr1 A / T 16766849 4 chr1 C / T 16876131 5 chr2 T / A 1449439 6 chr2 G / A 6047960 7 chr2 T / C 11792541 8 chr2 T / C 12993894 9 chr2 G / T 14753388 10 chr3 T / G 2458974 11 chr3 T / C 3615656 12 chr3 A / T 4138524 13 chr3 C / T 4176564 14 chr3 T / A 9155833 15 chr3 T / C 12232585 16 chr4 T / C 2886675 17 chr4 G / C 3608977 18 chr5 C / G 797202 19 chr5 G / A 1087235 20 chr5 A / C 3945498 21 chr5 G / A 4679588 22 chr5 G / A 10293664 23 chr5 G / C 10440102 24 chr5 T / A 11017191 25 chr5 C / A 11234246 26 chr5 T / A 11336507 27 chr5 G / C 11678375 28 chr5 C / T 11740440 29 chr5 G / T 12496016 30 chr6 A / T 3046896 31 chr6 A / G 7343286 32 chr7 A / C 7789035 33 chr8 G / C 5315740 34 chr8 T / C 12539071 35 chr9 C / G 4677174 36 chr9 T / G 6717585 37 chr9 A / G 9086655 38 chr9 C / T 9602230 39 chr9 A / G 9908303 40 chr9 C / T 12133358 41 chr9 G / T 14260481 42 chr10 A / G 1795421 43 chr10 A / C 3112444 44 chr10 G / T 3376971 45 chr10 G / A 6481666 46 chr10 A / C 7355867 47 chr10 A / G 7571691 48 chr10 C / A 7869501 49 chr10 A / G 7887601 50 chr10 C / T 15676925 51 chr10 C / G 16421439 52 chr10 A / G 16799434 53 chr10 G / A 17442307 54 chr10 C / A 19875781 55 chr10 A / T 19890964 56 chr11 C / T 4568981 57 chr11 T / C 9019089 58 chr11 C / T 12022564 59 chr12 T / C 2562320 60 chr12 T / A 3210543 61 chr12 T / C 3622383 62 chr13 C / T 8159487 63 chr13 T / C 11867192 64 chr13 A / G 13166742 65 chr13 T / G 14354150 66 chr14 C / T 5509421 67 chr14 C / A 6344930 68 chr14 G / T 19350623 69 chr15 A / G 8888342 70 chr17 A / T 2157608 71 chr17 G / T 11412020 72 chr18 G / A 5648593 73 chr20 T / G 4099423 74 chr20 G / T 4443386 75 chr20 A / T 4810100 76 chr20 C / G 5550447 77 chr20 C / T 6290104 78 chr20 C / G 8725304 79 chr20 G / T 9543195 80 chr22 A / G 9616602 81 chr22 T / C 12450621 82 chr23 G / A 5264616 83 chr23 T / G 7741418 84 chr23 G / C 13813480 85 chr24 A / G 10661457 86 chr25 C / G 6015449 87 chr25 A / G 7065406 88 chr26 G / C 1102503 89 chr26 A / G 1984131 90 chr26 G / A 12443874 91 chr26 T / A 12858134 92 chr26 A / G 14611223 93 chr26 T / G 15144360 94 chr27 C / T 1192175 95 chr27 G / C 8383657 96 chr28 T / C 3489096 97 chr28 A / G 4094329 98 chr28 A / G 10456006 99 chr28 A / G 11792708 100 chr28 C / G 13452278 101 chr28 G / C 13500082 102 chr28 C / T 14795793 103 chr29 C / A 7892530 104 chr29 A / T 8587895 105 chr29 G / C 9565045 106 chr29 A / C 9992068 107 chr29 A / G 12823258 108 chr29 A / T 13068794 109 chr30 A / T 8866843 110 chr30 T / G 9298070 111 chr30 C / T 9990589 112 chr30 T / C 11548043 113 chr31 A / G 1062805 114 chr31 A / C 1098316 115 chr31 G / A 1240759 116 chr31 G / T 1619992 117 chr31 C / T 2675443 118 chr31 G / A 3219688 119 chr31 C / A 6465152 120 chr31 T / C 11119549 121 chr31 G / T 15436077 122 chr32 T / A 443711 123 chr32 A / G 6444806 124 chr32 A / G 7095543 125 chr32 A / T 8795951 126 chr32 C / T 10280521 127 chr32 T / G 11253310 128 chr32 C / A 14611674 129 chr32 G / A 15741415 130 chr32 T / C 25824965

[0052] (2) Further utilize 130 SNPs to construct the DNA fingerprint map of the core germplasm set ( Figure 2 The R script was used to perform pairwise alignments of each locus in 91 samples and construct a matrix of differential loci between samples.

[0053] (3) The results of the inter-sample differential loci matrix show that only one pair of samples has 2 differential loci, only one pair of samples has 3 differential loci, and the number of differential loci among the remaining samples is 8 or more. This indicates that all materials in the core germplasm set can be completely distinguished, demonstrating a good identification ability for kelp germplasm materials. An example of the sample with the fewest inter-sample differential loci is as follows:

[0054] F21 and M20 both originated from the same strain, CKH-strain 2, and had the most similar genetic backgrounds. The F21 and M20 samples could be successfully distinguished using both the 50th SNP site located at sequence SEQ ID NO: 65 and the 50th SNP site located at sequence SEQ ID NO: 130 (Table 4).

[0055] Table 4: SNP locus information used to distinguish core germplasm materials F21 and M20

[0056] SNP locus F21 M20 chr13-14354150 (SEQ ID NO:65) T G chr32-25824965 (SEQ ID NO:130) C T

[0057] Therefore, the 130 SNP loci mentioned above can be used to achieve accurate identification of kelp core germplasm.

[0058] Example 3: Strain identification based on kelp core SNPs

[0059] (1) Both JLH-strain 1 and JLH-strain 6 are cultivated strains bred from hybridization of kelp (S. japonica) and longissima (S. longissima) and after multiple generations of self-pollination, and have a close kinship. Among them, kelp of JLH-strain 1 has the characteristics of longer thallus length and heat resistance, while kelp of JLH-strain 6 has the characteristic of wider thallus width.

[0060] (2) The core germplasm set contains 3 JLH-line 1 germplasm samples and 3 JLH-line 6 germplasm samples. Additionally, 7 JLH-line 1 germplasm samples and 9 JLH-line 6 germplasm samples were randomly selected from the germplasm bank, and the genotypes of the 130 core SNP loci obtained in Example 2 were acquired from these non-core germplasm samples. Statistical results showed that there were 12 stable SNP differential loci between JLH-line 1 and JLH-line 6; therefore, these differential SNPs could be used to successfully distinguish the two lines (Table 5).

[0061] Table 5: SNP locus information used to distinguish between JLH-strain 1 and JLH-strain 6

[0062]

[0063] In summary, this invention develops and screens SNP markers from representative gametophyte germplasm from kelp germplasm, constructing a core kelp germplasm with complete allele coverage for genetic diversity. The number of core germplasm samples accounts for 40% of the initial germplasm, which can significantly improve the efficiency of germplasm bank preservation and management. This invention screens 130 core SNP markers, which can not only achieve accurate identification of 91 core kelp gametophyte germplasm materials, but also be used for the identification of different kelp strains.

Claims

1. A genome-wide SNP marker from kelp, characterized in that, The SNP marker is located at the 50th position of any sequence in SEQ ID NO: 1-130.

2. The application of the SNP marker as described in claim 1 in the identification of kelp germplasm.

3. A method for identifying core germplasm samples of kelp, characterized in that, The method involves using one or more of the SNP markers described in claim 1 for identification.

4. The method as described in claim 3, characterized in that, The aforementioned kelp core germplasm samples are a set of 91 core samples constructed based on genetic diversity and germplasm resource background information.

5. The method as described in claim 3, characterized in that, All samples underwent pairwise differential analysis to ensure that there were at least 2 different loci between any two samples.

6. A method for identifying kelp strains, characterized in that, The method involves using 12 of the SNP markers described in claim 1 for identification.

7. The method as described in claim 6, characterized in that, The kelp population mentioned is the JLH cultivar, a hybrid of kelp and long kelp.

8. The method as described in claim 6, characterized in that, The SNP markers include one or more of the following SNP sites: 1) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:2; 2) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:6; 3) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:7; 4) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:19; 5) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:81; 6) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:92; 7) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:94; 8) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:102; 9) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:103; 10) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:104; 11) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:107; 12) The SNP site located at position 50 of the nucleic acid fragment with sequence SEQ ID NO:

108.

9. A method for constructing kelp core germplasm based on SNP molecular markers, characterized in that, The method includes the following steps: 1) Obtain kelp gametophyte germplasm resource samples with complete and accurate information sources, extract genomic DNA from all samples, and perform DNA sample testing, library construction, whole genome resequencing, and quality control to obtain high-quality sequencing data; 2) Using the resequencing data obtained in step 1), SNP sites are detected by aligning to the reference genome, and high-quality SNP datasets are detected and filtered out. 3) Using the high-quality SNP dataset obtained in step 2) as genetic information data, construct core germplasm sets with different proportions using Core Hunter3 software; 4) For the core germplasm sets with different proportions in step 3), analyze their genetic diversity parameters and combine them with germplasm resource background information to finally determine the core germplasm of kelp; 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.

10. The method as described in claim 8, characterized in that, The SNP tag set mentioned above is the SNP tag set as described in claim 1.

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