SNP (single nucleotide polymorphism) molecular marker combination for constructing siberian wildrye DNA (deoxyribonucleic acid) fingerprint spectrum and application

Abstract

The invention relates to the technical field of molecular biology of siberian wildrye, and particularly discloses an SNP (Single Nucleotide Polymorphism) molecular marker combination for constructing a siberian wildrye DNA (Deoxyribose Nucleic Acid) fingerprint spectrum and application. The SNP molecular marker combination for constructing the siberian wildrye DNA fingerprint spectrum is composed of 52 SNP molecular markers, and the SNP molecular marker combination has high specificity and resolution, and can reveal the real level of genetic diversity of siberian wildrye; the SNP molecular marker combination is used for establishing a siberian wildrye DNA fingerprint database, DNA fingerprint information of different siberian wildrye germplasm can be correctly reflected, resource or variety identification can be carried out on siberian wildrye, the detection result is accurate and efficient, 90 siberian wildrye germplasm resources are effectively distinguished for the first time, the identification capacity is high, and technical support is provided for siberian wildrye germplasm resource protection. The SNP marker and the application method thereof provided by the invention have the advantages of high throughput, accuracy, low cost, simplicity in operation and the like, and have a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology of Elymus sibiricus, and particularly relates to a SNP molecular marker combination and application for constructing a DNA fingerprint of Elymus sibiricus. Background Art

[0002] Elymus sibiricus L. is the model species of the genus Elymus L., Triticeae, Poaceae. It is a perennial, allotetraploid grass (2n=4x=28, StStHH), widely distributed in Eurasia, and can form dominant and community-building species in meadow steppe communities. Elymus sibiricus not only has good forage quality, but also has strong adaptability and excellent disease resistance to adverse environments such as high altitude, cold, drought, and salinity. It is widely used in the restoration of degraded grassland vegetation and the establishment of high-yield artificial grassland in the Qinghai-Tibet Plateau and other high-altitude areas in western China. It is also an important gene bank for improving wheat crops and forages. Due to its high seed production potential, Elymus sibiricus is one of the few native grass species in the Qinghai-Tibet Plateau that has achieved large-scale seed production and commercial use.

[0003] The wild resources of Siberian barley are widely distributed and have diverse habitats, including alpine meadows, forest meadows, shrubs, alpine canyons, river valley gravel beaches, etc. at an altitude of 1500 to 4900 meters. Affected by the climatic factors of different habitats, there are obvious phenotypic and genetic differences between wild Siberian barley populations, which provides rich genetic resources and a diverse selection basis for the development and utilization of Siberian barley germplasm, but also increases the difficulty of effective protection of Siberian barley resources. At present, the identification of Siberian barley resources mainly relies on phenotypic characteristics, but the morphological variation of wild Siberian barley germplasm is extensive, and its traits are often affected by environmental changes. In addition, it has many similarities in phenotype with species of the same genus such as Elymus nutans, and their geographical distribution areas also overlap, so it is difficult to accurately identify them by phenotype in the wild. Therefore, efficient and accurate resource identification is of great significance for the effective protection, rational utilization and intellectual property protection of Siberian barley genetic diversity.

[0004] DNA fingerprints are composed of a small number of highly representative markers, which can essentially reflect the differences between biological individuals and distinguish different individuals within the same species. They have high individual specificity and stable reliability. They are an effective means to conveniently, quickly, accurately and stably identify germplasm resources and varieties, and are widely used in crop germplasm resource research. At present, the commonly used molecular markers used to construct fingerprints are RFLP, RAPD, AFLP, ISSR, SSR, SRAP, etc. These methods are time-consuming, inefficient and not highly compatible. It is urgent to establish a set of high-throughput, high-efficiency, high-compatibility, easy-to-operate, and low-cost molecular technologies to promote the protection, research and utilization of Elymus sibiricus genetic resources.

[0005] SNP markers, or single nucleotide polymorphism markers, refer to genetic markers formed by the variation of a single nucleotide in the genome, including substitution, transversion, deletion and insertion. They are the simplest and most common form of polymorphism in the genome. SNP markers are numerous and widely distributed, with high genetic stability, strong representativeness, easier genotyping, and fast and efficient detection. They are widely used in the construction of core germplasms and fingerprint maps of crops such as rice, corn, sorghum, tea, and soybeans, providing important resources for crop breeding, genome research, and genetics research. They also play an important role in crop variety identification, purity determination, and variety rights protection. However, to date, there has been no report on the construction of a DNA fingerprint map of Siberia sibiricum based on SNP markers. Therefore, using SNP sites to identify Siberia sibiricum germplasm and constructing a fingerprint database of Siberia sibiricum based on core SNP sites is very important for the protection of genetic diversity of Siberia sibiricum, the rational use of germplasm resources, and the protection of intellectual property rights. Summary of the invention

[0006] The purpose of the present invention is to provide a SNP molecular marker combination and application for constructing a DNA fingerprint of Elymus sibiricus, aiming to provide a stable and efficient molecular means for the effective protection, rational utilization and intellectual property protection of Elymus sibiricus germplasm resources.

[0007] To achieve the above object, the present invention provides a SNP molecular marker combination for constructing a DNA fingerprint of Elymus sibiricus. The SNP molecular marker combination consists of 52 SNP molecular markers numbered Es-1 to Es-52. The specific information is shown in Table 1.

[0008] Table 1 Information on 52 SNP molecular markers in Elymus sibiricus

[0009]

[0010]

[0011] On the other hand, the present invention also provides the use of the above-mentioned SNP molecular marker combination in any of the following aspects:

[0012] (1) Used for the construction of DNA fingerprint of Elymus sibiricus;

[0013] (2) Used for genotyping of Elymus sibiricus;

[0014] (3) Used for identification of Elymus sibiricus germplasm resources and varieties.

[0015] On the other hand, the present invention also provides a method for constructing a DNA fingerprint of Elymus sibiricus germplasm resources or varieties, comprising the following steps:

[0016] S1. Extracting DNA from Elymus sibiricus germplasm resources or varieties for constructing DNA fingerprints, and detecting the genotypes of the 52 SNP sites;

[0017] S2. The genotypes of each Elymus sibiricum germplasm resource or variety at the 52 SNP sites are arranged in combination according to the site number sequence to form a specific sequence, which is the DNA fingerprint map of the Elymus sibiricum germplasm resource or variety.

[0018] Preferably, in the above method for constructing a DNA fingerprint of Elymus sibiricus germplasm resources or varieties, the specific sequence obtained in step S2 is encoded, and a QR code or barcode recognizable by a device terminal is constructed to intelligently identify and manage Elymus sibiricus germplasm resources or varieties.

[0019] On the other hand, the present invention also provides a DNA fingerprint database of Elymus sibiricus resources constructed by the above construction method.

[0020] Compared with the existing technology, the present invention has the following beneficial effects:

[0021] The present invention is used to construct a SNP molecular marker combination for DNA fingerprinting of Siberia sibiricum, which is composed of 52 SNP molecular markers, has high specificity and resolution, and can reveal the true level of genetic diversity of Siberia sibiricum; the DNA fingerprint library is established by using the SNP molecular marker combination, which can correctly reflect the DNA fingerprint information of different Siberia sibiricum resources or varieties, and can identify Siberia sibiricum resources or varieties, effectively distinguishing 90 Siberia sibiricum germplasm resources for the first time, with strong identification ability, providing technical support for the protection of Siberia sibiricum germplasm resources. The SNP molecular marker combination and its application method provided by the present invention have the advantages of high throughput, precision, high efficiency, simple operation, low cost, etc., and have a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a cluster diagram of 90 Elymus sibiricum germplasm resources of the present invention;

[0023] Figure 2 The minor allele frequency (MAF) and polymorphism information content (PIC) values ​​of 90 tested Elymus sibiricus germplasms based on 338 candidate SNP markers in Example 1 of the present invention;

[0024] Figure 3 The observed heterozygosity (Ho) of 90 tested Elymus sibiricus germplasms based on 338 candidate SNP markers in Example 1 of the present invention;

[0025] Figure 4 Nei's diversity index of 90 tested Elymus sibiricum germplasms based on 338 candidate SNP markers in Example 1 of the present invention;

[0026] Figure 5 This is a phylogenetic tree of 90 tested Elymus sibiricus germplasms based on 338 SNP markers in Example 1 of the present invention, where QTP represents the Qinghai-Tibet Plateau population, NE represents the Northeast population, NW represents the Northwest population, and NC represents the North China population;

[0027] Figure 6 This is the location map of 52 SNP sites on chromosomes;

[0028] Figure 7 The genotyping results of 90 tested Elymus sibiricus germplasms based on 52 SNP markers in Example 2 of the present invention are shown below: each row represents a SNP genotype, each column represents a material, and yellow, green, blue and purple represent nucleotides C / C, A / A, T / T and G / G, respectively. Missing data are shown in gray, and heterozygous sites are shown in white;

[0029] Figure 8 This is a population structure analysis of 90 samples of Elymus sibiricum based on 52 core SNP markers. From top to bottom, the figure shows the phylogenetic tree of 90 Elymus sibiricum samples based on 52 core SNP markers, sample names, and population structures of 90 Elymus sibiricum accessions at different K values. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0031] Example 1 SNP sites used to construct DNA fingerprint of Elymus sibiricus

[0032] 1. Test materials

[0033] The present invention uses 90 Elymus sibiricum germplasms as samples, and the germplasm resource information is shown in Table 4. These 90 Elymus sibiricum germplasms come from the Qinghai-Tibet Plateau, Northeast China, Northwest China and North China, Russia and Mongolia, covering the main distribution areas of wild Elymus sibiricum, representing a wide range of Elymus sibiricum genetic diversity. Figure 1 .

[0034] 2. Extraction of genomic DNA from the tested Elymus sibiricum germplasm

[0035] The genomic DNA of the tested E. sibiricum germplasm was extracted using a plant DNA extraction kit (DP350, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the DNA concentration and purity were detected using a NanoDrop2000 UV spectrophotometer (Thermo Fisher Scientific, MA, USA).

[0036] 3. SNP acquisition

[0037] The 90 genomic DNA samples extracted from step 2 were used for whole genome resequencing at Biomarker Biotechnology Co., Ltd. (Beijing) (average sequencing depth was about 11 X ), and used the software BWA (Burrows-WheelerAligner) to align it to the reference genome of Elymus sibiricus, Chuancao 2 (assembled by the inventor's research group: https: / / www.biorxiv.org / content / 10.1101 / 2024.04.17.589894v1), and identified 80,148,422 high-quality SNPs through GATK (GenomeAnalysis Toolkit).

[0038] 4. Candidate SNP marker screening and effectiveness evaluation

[0039] The screening principles are as follows:

[0040] (1) Specificity: To ensure that the selected SNP markers have high specificity, 200 bp of sequence upstream and downstream of each SNP were extracted, totaling 401 bp. Then, BLAST (Basic Local Alignment Search Tool) was performed to retain only those SNPs that could be uniquely aligned to the reference genome.

[0041] (2) Uniform distribution and high quality: Based on the premise that SNP markers are evenly distributed on the 14 chromosomes of E. sibiricum, SNPs with no missing genotype data were retained, and SNPs with minor allele frequency (MAF) lower than 20% were eliminated.

[0042] (3) Polymorphic Information Content (PIC): The Polymorphic Information Content (PIC) value of the SNP locus was calculated using a Perl script, and loci with a PIC value less than 0.35 were eliminated.

[0043] (4) Hardy-Weinberg equilibrium: Use VCFtools software and set the parameters --max-missing 1 --maf 0.2 --hwe 0.01 to retain sites with p values ​​greater than 0.01 in the Hardy-Weinberg test.

[0044] (5) Uniqueness: Perl script was used to screen out SNP sites with no other site mutations within 100 bp before and after the marker.

[0045] Based on the above principles, 338 SNP loci were finally obtained as candidate markers. Genetic diversity analysis of the 338 candidate markers showed that the MAF and PIC values ​​of the 338 SNP loci varied in the range of 0.35-0.50 and 0.351-0.375, respectively, with average values ​​of 0.421 and 0.367, respectively (see Figure 2 ); Ho (Observed Heterozygosity) is 0.333~0.644, with an average of 0.484 (see Figure 3 ); the average values ​​of Nei genetic diversity index and Shannon-Wiener index were 0.486 and 0.676 respectively (see Figure 4 ). These results indicated that the 338 candidate markers showed strong polymorphism.

[0046] The accuracy and effectiveness of the candidate markers were evaluated by using the above 338 candidate markers to analyze the population structure of 90 tested Elymus sibiricus germplasms. Figure 5 ), found that except for a few materials with close geographical distribution that could not be distinguished according to geographical origin, this may be related to the convergent genetic variation shown by the environmental similarity of these materials and the existence of gene flow between populations. The 338 candidate SNP markers can effectively distinguish most of the old wheat populations from different geographical origins, especially the Qinghai-Tibet Plateau population with obvious differences in geographical environment and the non-Qinghai-Tibet Plateau population. This is basically consistent with the results based on whole genome SNP clustering ( Figure 1 ), indicating that the 338 candidate SNP markers could effectively reflect the genetic similarity among these germplasms.

[0047] 5. Core SNP marker screening and effectiveness evaluation

[0048] Based on the genetic diversity information of the 338 candidate SNPs obtained above, we further screened SNP loci that were evenly distributed on the 14 chromosomes, had no missing genotype data, and were detected in as many individuals as possible, and finally obtained 52 core SNP markers. The specific information is shown in Table 1, and the positions of the 52 SNP markers on the 14 chromosomes of E. sibiricum are shown in Figure 6 .

[0049] Using the above 52 core SNP markers, 90 tested Elymus sibiricum samples were compared in pairs, and the results were able to distinguish each Elymus sibiricum germplasm ( Figure 7 ). In addition, the population structure of 90 samples of Elymus sibiricum was analyzed by using the above 52 core SNP markers, which can accurately distinguish the germplasm from the Qinghai-Tibet Plateau population from the germplasm from the non-Qinghai-Tibet Plateau population, indicating that the above 52 core SNP markers can effectively represent the 338 candidate SNP markers ( Figure 8 ).

[0050] Example 2 Genotyping and fingerprinting of Elymus sibiricus germplasm resources

[0051] Fingerprint construction: DNA of leaf samples of 90 E. sibiricum germplasms for constructing DNA fingerprints were extracted according to the method of step 2 in Example 1, and whole genome resequencing was completed at Biomarker Biotechnology Co., Ltd. (Beijing) to obtain the genotypes of the above 52 SNP sites of 90 E. sibiricum germplasm resource samples; according to the genotyping results, the genotypes of each E. sibiricum germplasm at the 52 SNP sites were combined and arranged in the order of site numbers to form a specific sequence, which is the DNA fingerprint of the E. sibiricum germplasm resources, as shown in Table 4. The typing results of the 90 test materials are shown in Figure 7 , where each row represents a SNP genotype, each column represents a material, yellow, green, blue and purple represent nucleotides C / C, A / A, T / T and G / G, respectively, missing data are displayed in gray, and heterozygous sites are displayed in white. The online software Caoliiaoerweima (http: / / cli.im / ) was used to encode the genotype data (specific sequence) of 52 core SNPs of 90 Siberian sibiricum germplasms, and a two-dimensional barcode was constructed for each Siberian sibiricum material, as shown in Table 4. The samples with a valid SNP data ratio of ≥0.80 were considered valid samples, and the 52 SNP markers could effectively distinguish the 90 Siberian sibiricum germplasms.

[0052] Table 4 Sources and DNA fingerprints of 90 Elymus sibiricus germplasms

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Example 3 A method for identifying a germplasm or variety of Elymus sibiricus

[0061] The DNA of the tested E. sibiricum germplasm is extracted, and the genotype of the above 52 SNP sites of the genomic DNA of the tested E. sibiricum germplasm is detected by the whole genome resequencing method, and compared with the E. sibiricum DNA fingerprint library in Example 2 to identify the E. sibiricum germplasm. The percentage of the number of effective sites that the tested sample and the standard sample have in common with the number of effective sites of the standard sample is used to identify whether the tested sample is a standard variety or a similar variety or a suspected variety in the E. sibiricum DNA fingerprint library in Example 2. If the number of effective sites of the tested E. sibiricum germplasm and a certain standard E. sibiricum germplasm coincide with each other at more than 98%, the tested E. sibiricum germplasm and the standard E. sibiricum germplasm are or are suspected to be the same E. sibiricum germplasm. If the number of effective sites of the tested E. sibiricum germplasm and a certain standard E. sibiricum germplasm coincide with each other at 98% or less, the tested E. sibiricum germplasm and the standard E. sibiricum germplasm belong to different E. sibiricum germplasms, and the lower the coincidence rate, the more distant the genetic relationship.

[0062] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A combination of SNP molecular markers for constructing a DNA fingerprint of Elymus sibiricus, characterized in that: The SNP molecular marker combination consists of 52 SNP molecular markers, numbered Es-1 to Es-52, and the specific information is shown in Table 1; Table 1 Information on 52 SNP molecular markers in Elymus sibiricus 2. The use of the SNP molecular marker combination according to claim 1, characterized in that: Use of the molecular marker combination according to claim 1 in any of the following aspects: (1) Used for the construction of DNA fingerprint of Elymus sibiricus; (2) Used for genotyping of Elymus sibiricus; (3) Used for identification of Elymus sibiricus germplasm resources and varieties.

3. A DNA fingerprint database of Elymus sibiricus germplasm resources, characterized in that: The method for constructing the fingerprint database is as follows: Step 1. extracting DNA of Elymus sibiricum germplasm resources or varieties for constructing DNA fingerprints, and detecting the genotypes of the 52 SNP molecular markers described in claim 1; Step 2. The genotypes of each Siberian Elaeagnus germplasm resource or variety at the 52 SNP sites are combined and arranged in the order of site numbers to form a specific sequence, which is the DNA fingerprint of the Siberian Elaeagnus germplasm resource or variety.

4. An intelligent identification code for the DNA of Elymus sibiricus germplasm resources, characterized in that: The method for constructing the intelligent identification code is as follows: Step 1. extracting DNA of Elymus sibiricum germplasm resources or varieties for constructing DNA fingerprints, and detecting the genotypes of the 52 SNP molecular markers described in claim 1; Step 2. Arrange the genotypes of each Elymus sibiricum germplasm resource or variety at the 52 SNP sites in the order of site numbers to form a specific sequence, which is the DNA fingerprint of the Elymus sibiricum germplasm resource or variety; Step 3. Encode the specific sequence obtained in step S2 and construct a QR code or barcode that can be recognized by the device terminal to intelligently identify and manage the germplasm resources or varieties of Elymus sibiricus.

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