Polymorphic primer of Chinese hippophae rhamnoides genome SSR molecular marker and application of polymorphic primer

Abstract

The invention belongs to the technical field of molecular biology, and provides polymorphic primers of Chinese hippophae rhamnoides genome SSR molecular markers and application of the polymorphic primers. The SSR molecular marker is composed of 20 pairs of SSR molecular marker polymorphic primers, and the nucleotide sequences of the primers are as shown in SEQ ID NO.1-SEQ ID NO.40. The SSR molecular marker comprises 20 pairs of SSR molecular marker polymorphic primers. A large number of SSR sequences are mined by using transcriptomes, SSR capillary electrophoresis technology is combined, SSR loci with high measurement indexes Na value, I value and PIC value of genetic difference and front 20 of the sequence are screened, and Chinese sea-buckthorn molecular identity information is made and applied to Chinese sea-buckthorn geographic source identification. The SSR molecular marker is good in polymorphism primer specificity and high in polymorphism, and when the SSR molecular marker is applied to Chinese hippophae rhamnoides population genetic structure and resource genetic diversity analysis, the diversity and genetic structure of hippophae rhamnoides genetic level in China can be revealed more comprehensively, and a foundation is laid for formulating a genetic protection strategy.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to polymorphic primers for SSR molecular markers of the Chinese sea buckthorn genome and their applications. Background Technology

[0002] Sea buckthorn ( Hippophae rhamnoides L. Sea buckthorn (Hippophae rhamnoides) is a perennial deciduous shrub or small tree, a light-loving, water-tolerant, drought-tolerant, and highly vigorous small-berry plant. my country has the richest resources of sea buckthorn, mainly distributed in North China, Northwest China, and Southwest China. Shanxi is a major producer of sea buckthorn in China. Hippophae rhamnoides subsp. sinensis China is a resource-rich province and one of the origins of sea buckthorn. Currently, the province has 6.3 million mu of sea buckthorn forest, including 5 million mu of wild sea buckthorn and 1.3 million mu of planted sea buckthorn. Sea buckthorn is rich in vitamins, polysaccharides, proteins, amino acids, trace elements, and bioactive substances such as flavonoids, steroidal compounds, proanthocyanidins, triterpenes, and serotonin. It possesses both medicinal and edible properties and has received widespread attention in the development and research of health products and food. Currently, 19 counties and cities in the province are located along the Yellow River, with a total sea buckthorn area of ​​433,300 mu, accounting for 9.6% of the province's total sea buckthorn area. Of this, 249,800 mu are natural forests, all distributed in the Lüliang Mountains, mainly used as an important tree species for soil and water conservation and windbreak and sand fixation. The evaluation of the genetic diversity of sea buckthorn in China is of great significance for the development and utilization of sea buckthorn in the Yellow River Basin.

[0003] The middle and upper reaches of the Yellow River are the richest region for sea buckthorn germplasm resources, and also the center of taxonomic differentiation, primitive taxa, and origin of the genus Hippophae. Sea buckthorn has significant ecological, economic, and social benefits, thus its resources have received increasing attention and application. However, research on the genetic diversity of sea buckthorn in the middle and upper reaches of the Yellow River has not been reported.

[0004] In recent years, with my country's continued focus on the value of sea buckthorn, hybridization breeding, molecular breeding and other technologies have been widely used in the selection of sea buckthorn strains. In addition, with the continuous introduction of excellent sea buckthorn germplasm resources from abroad, a large number of closely related strains with varying biological characteristics, ecological adaptability and quality traits have emerged. This has brought challenges to the identification and approval of superior sea buckthorn varieties, the preservation, production and management of seedlings, the development of the sea buckthorn industry and the selection of soil and water conservation.

[0005] Given the above, traditional morphological and cytological identification methods are no longer sufficient for the rapid and accurate identification and research of sea buckthorn strains. Existing literature reports the following: genetic diversity of sea buckthorn was analyzed by screening grape SSR primers; SRAP was used to optimize the PCR program and screen primers for sea buckthorn; however, both of these markers have limitations in primer selection and combination; RAPD was used to identify sea buckthorn strains, but the results were unstable and had low reproducibility due to the lack of DNA probes in RAPD. Summary of the Invention

[0006] This invention provides polymorphic primers for SSR molecular markers of the Chinese sea buckthorn genome and their applications.

[0007] This invention is achieved by the following technical solution: polymorphic primers for SSR molecular markers in the Chinese sea buckthorn genome, consisting of the following 20 pairs of polymorphic primers for SSR molecular markers, with the primer nucleotide sequences as follows: The forward primer for SZ01 is shown in SEQ ID NO. 1; the reverse primer is shown in SEQ ID NO. 2. The forward primer for SZ03 is shown in SEQ ID NO. 3; the reverse primer is shown in SEQ ID NO. 4. The forward primer for SZ08 is shown in SEQ ID NO. 5; the reverse primer is shown in SEQ ID NO. 6. The forward primer for SZ16 is shown in SEQ ID NO. 7; the reverse primer is shown in SEQ ID NO. 8. The forward primer for SZ34 is shown in SEQ ID NO. 9; the reverse primer is shown in SEQ ID NO. 10. The forward primer for SZ56 is shown in SEQ ID NO. 11; the reverse primer is shown in SEQ ID NO. 12. The forward primer for SZ57 is shown in SEQ ID NO. 13; the reverse primer is shown in SEQ ID NO. 14. The forward primer for SZ67 is shown in SEQ ID NO. 15; the reverse primer is shown in SEQ ID NO. 16. The forward primer for SZ68 is shown in SEQ ID NO. 17; the reverse primer is shown in SEQ ID NO. 18. The forward primer for SZ79 is shown in SEQ ID NO. 19; the reverse primer is shown in SEQ ID NO. 20. The forward primer for SZ88 is shown in SEQ ID NO. 21; the reverse primer is shown in SEQ ID NO. 22. The forward primer for SZ95 is shown in SEQ ID NO. 23; the reverse primer is shown in SEQ ID NO. 24. The forward primer for SZ97 is shown in SEQ ID NO. 25; the reverse primer is shown in SEQ ID NO. 26. The forward primer for SZ100 is shown in SEQ ID NO. 27; the reverse primer is shown in SEQ ID NO. 28. The forward primer for SZ102 is shown in SEQ ID NO. 29; the reverse primer is shown in SEQ ID NO. 30. The forward primer for SZ110 is shown in SEQ ID NO. 31; the reverse primer is shown in SEQ ID NO. 32. The forward primer for SZ111 is shown in SEQ ID NO. 33; the reverse primer is shown in SEQ ID NO. 34. The forward primer for SZ114 is shown in SEQ ID NO. 35; the reverse primer is shown in SEQ ID NO. 36. The forward primer for SZ124 is shown in SEQ ID NO. 37; the reverse primer is shown in SEQ ID NO. 38. The forward primer for SZ126 is shown in SEQ ID NO. 39; the reverse primer is shown in SEQ ID NO. 40.

[0008] Furthermore, the 5' end of the forward primer is modified with M13, the sequence of which is 5'-TGTAAAACGACGGCCAGT-3'; a fluorescent group is attached to the 3' end. The fluorescent group is one of FAM, HEX, ROX, and NED.

[0009] This invention also provides the application of the polymorphic primers for the SSR molecular markers of the Chinese sea buckthorn genome in the analysis of the genetic structure and genetic diversity of Chinese sea buckthorn populations. Furthermore, this invention provides the application of the polymorphic primers for the SSR molecular markers of the Chinese sea buckthorn genome in marker-assisted breeding of Chinese sea buckthorn. Finally, this invention provides the application of the polymorphic primers for the SSR molecular markers of the Chinese sea buckthorn genome as molecular identity information for identifying the geographical origin of Chinese sea buckthorn.

[0010] Furthermore, this includes the following steps: 1) Extract genomic DNA from Chinese sea buckthorn; 2) The genomic DNA extracted in step 1) was amplified by PCR using the primers shown in SEQ ID NO. 1-SEQ ID NO. 40 above to obtain the amplification product; 3) Perform polyacrylamide gel electrophoresis and / or capillary electrophoresis on the amplification products obtained in step 2). 4) Data analysis of SSR locus polymorphism in the nuclear genome to conduct genetic structure and genetic diversity analysis of Chinese sea buckthorn populations and molecular marker-assisted breeding.

[0011] The PCR amplification system consisted of: 17.5 μL of 2×Taq PCR premixed reagent II, 10 μmol·L⁻¹ -1 1.0 μL each of forward and reverse primers, 10 μmol·L⁻¹ -1 0.5 μL of M13 fluorescently labeled primers, 0.8 μL of genomic DNA, and 4.2 μL of ddH2O were used. The total reaction volume was 25 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, decreasing by 1℃ per cycle, and 72℃ extension for 30 s, for a total of 6 cycles; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0012] This invention sequenced the transcriptome of *Hippophae rhamnoides* in China using the Illumina Novaseq™ 6000 sequencing platform. A total of 2,149,567,504 high-quality reads were obtained, which were assembled using de novo sequencing to yield 222,626 transcripts with an average length of 812 bp. A total of 85,380 unigenes were annotated, with the most annotated unigenes (53,578) in the eggNOG database, followed by the Nr database (47,063). 30,719 unigenes were annotated in the KEGG database, covering 126 KEGG standard metabolic pathways.

[0013] Studies have shown that the SSR loci in the transcriptome of Chinese sea buckthorn have a relatively high frequency and short average distribution distance, indicating that the number and types of SSR loci in the Chinese sea buckthorn transcriptome are abundant, suggesting that Chinese sea buckthorn has high polymorphic potential. The frequency of SSR loci decreases overall with increasing repeat number. Therefore, most of the SSR loci in the sequenced Chinese sea buckthorn transcriptome have polymorphic potential and low mutation frequency, making them suitable for the design and development of highly polymorphic Chinese sea buckthorn SSR primers. The high frequency and close distribution of SSR loci in the Chinese sea buckthorn transcriptome, along with the rich variety of repeat types and repeat motifs, provide a solid foundation for the development of primers with polymorphic potential, thus paving the way for the implementation of this invention.

[0014] This invention utilizes transcriptome mining to extract a large number of SSR sequences, and combines SSR capillary electrophoresis technology to screen out the top 20 SSR loci with high Na, I, and PIC values, which are indicators of genetic difference. This information is used to create molecular identity information for Chinese sea buckthorn and is applied to the identification of the geographical origin of Chinese sea buckthorn.

[0015] Based on the research results of this invention, it was found that the genetic relationship of the natural distribution of sea buckthorn in China presents the following geographical distribution pattern: along the Yellow River Basin, the genetic distance between populations distributed from south to north shows an increasing trend, that is, the kinship gradually becomes more distant with increasing latitude (from south to north).

[0016] The SRR molecular markers described in this invention are abundant, covering the entire genome, and exhibit high polymorphism. This invention employs capillary electrophoresis, which, compared to traditional gel electrophoresis, offers higher resolution, accuracy, efficiency, and speed, and enables automated operation. The Chinese sea buckthorn population tested in this invention has broad coverage, enabling a more comprehensive understanding of the genetic diversity and genetic structure of Chinese sea buckthorn. Attached Figure Description

[0017] Figure 1 Electrophoresis diagram for partial labeling and screening of SSR primers; Figure 2 UPGMA clustering tree of Hippophae rhamnoides in China constructed for genetic distance; Figure 3 Linear graphs showing the K-values ​​and ΔK-values ​​for 95 sea buckthorn strains; Figure 4 For the structure diagram, K=7; Figure 5 The structure diagram has K=4. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0020] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0022] I. Experimental Materials and Instruments 1. Experimental Materials: Wild Chinese sea buckthorn leaves collected from Shanxi Province in 2022-2023 were used as sampling sites in six cities with relatively concentrated natural Chinese sea buckthorn forests: Datong, Shuozhou, Taiyuan, Lüliang, and Linfen. The sampling locations included Anze (2), Baode (4), Fangshan (5), Guangling (1), Hunyuan (2), Jixian (3), Jiaokou (6), Jingle (3), Kelan (7), Lanxian (2), Loufan (4), Ningwu (5), Pianguan (6), Puxian (6), Wenshui (7), Wuzhai (5), Xixian (7), and Xingxian (7). 6), 100 Chinese sea buckthorn strains including Youyu (7), Yuanping (3), Yunzhou District (2), Zhongyang (5), and Zuoyun (2); The experimental material was healthy leaves of Chinese sea buckthorn free from diseases and pests, with young leaves being preferred (referring to leaves located on the upper part of the branches of the sea buckthorn plant, which have just developed and have not yet fully unfolded or have just unfolded). Each sample was at least 5g, and 3 replicates were randomly selected. After sampling, the samples were placed in a self-sealing bag containing color-changing silica gel (the amount of color-changing silica gel should be enough to cover the sample), and then stored in an ultra-low temperature freezer at -80℃ for subsequent experiments.

[0023] 2. Experimental Instruments: DNA was extracted using a kit from Sangon Biotech (Shanghai) Co., Ltd. DNA quality, concentration, and purity were determined by agarose gel electrophoresis and a spectrophotometer (NanoDrop 2000, Thermo Scientific). The OD of the samples was measured. 260 / OD 280 The value should be between 1.7 and 1.9, and stored at -20℃.

[0024] II. Experimental Methods 1. Transcriptome SSR Development and Primer Design: Fresh young leaves of Chinese sea buckthorn were used for RNA extraction and cDNA library construction. After library quality control, Illumina NovaSeq 6000 (Illumina, USA) S4 kit was used for high-throughput sequencing on the Illumina Hiseq 2000 platform (Illumina, USA). The obtained sequences were assembled into independent gene sets using Trinity, i.e., a transcriptome assembled from EST sequences. MISA (http: / / pgrc.ipk-gatersleben.de / misa / misa.html, default parameters) was used to identify sea buckthorn EST-SSR sites, and primers were designed using Primer 3 (version 2.3.4). The 5' end of the original upstream primer was modified with M13 (5'-TGTAAAACGACGGCCAGT-3'), and then M13 adapters with fluorescent 3' ends (FAM, HEX, ROX, NED) were synthesized. The PCR amplification products were detected by sequence complementation.

[0025] 2. Genomic DNA Extraction and Detection: Genomic DNA was extracted from Chinese sea buckthorn leaves using a high-efficiency plant genomic DNA extraction kit (DP350, Tiangen Biotech Co., Ltd., Beijing, China). The obtained products were analyzed for DNA quality and concentration using 1% agarose gel electrophoresis and a Nanodrop 2000 micro-ultraviolet spectrophotometer (Thermo Scientific, USA). The DNA concentration of each sample was then diluted to 20 ng / μL with sterile ddH2O for later use.

[0026] 3. PCR reaction and capillary electrophoresis detection of products: Based on the SSR locus information obtained from transcriptome data analysis, 20 pairs of primers with good polymorphism were designed and screened using the SSR loci. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and a carboxyfluorescein (FAM) label was added to the 5' end of the upstream primer. The PCR reaction system consisted of 17.5 μL of 2×Taq PCR premixed reagent II and 10 μmol·L⁻¹. -1 1.0 μL each of forward and reverse primers, 10 μmol·L⁻¹ -1 0.5 μL of M13 fluorescently labeled primers, 0.8 μL of genomic DNA, and 4.2 μL of ddH2O were used. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s (decreasing by 1℃ per cycle), 72℃ extension for 30 s, for a total of 6 cycles; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0027] The PCR product was diluted 10-fold, and 1 μL was added to a 96-well plate. Then, 9 μL of a mixture of formamide and molecular weight internal standard GeneScan500 LIZ (Thermo Fisher Scientific, USA) at a volume ratio of 9.5:0.5 was added. The plate was incubated at 95℃ for 5 min, then rapidly cooled on ice for 10 min, centrifuged at 3000 rpm for 1 min, and the amplified PCR product was subjected to capillary electrophoresis on an ABI-3730XL gene analyzer (Applied Biosystems, Foster City, CA) to detect fluorescence signals and elution sites.

[0028] The transcriptome data of Chinese sea buckthorn obtained from leaf sequencing were de novo assembled, and then the obtained Unigenes sequences were searched for SSR sites using MISA software. The search was performed at least 5 times for each nucleotide repeat motif. Since the SSR sites of single nucleotide repeat motifs are rarely used in practical applications, they were not screened.

[0029] Genetic diversity parameters, namely allele count (Na), effective allele count (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and Shannon's information index, were calculated using POPGENE 1.32 software. Polymorphic information content (PIC) was calculated using the formula. The correlation between genetic distance and geographical distance was analyzed using GenALEx software, and molecular variance analysis was performed on Hippophae rhamnoides progeny within and between progeny. Cluster analysis was performed using NTSYS 2.1 software, and genetic structure analysis was performed using STRUCTURE 2.3.4 software. The genetic structure of Hippophae rhamnoides in the middle and upper reaches of the Yellow River was analyzed and comprehensively evaluated based on the above analytical results.

[0030] The format was converted using Convert (version 1.31), and the genetic diversity parameters (Na, Ne, Ho, He, PIC, I) of each primer pair were calculated using Popgene (version 1.32) and Cervus (version 3.0.7) to compare primer polymorphism. Pearson correlation analysis was performed using OriginPro 2010 (version 9.8.0.200). Genetic similarity coefficients were analyzed using NTSYSpc (version 2.10e), and UPGMA clustering dendrograms were plotted. The correlation between similarity coefficients was analyzed based on the MxComp function in NTSYSpc (version 2.10e) software to evaluate the quality of the phylogenetic dendrogram and analyze the phylogenetic relationships between strains. Finally, primer combinations were optimized to identify all strains, and the probability of identical fingerprint patterns was calculated by substituting the number of polymorphic sites of the selected primers into the formula P=1 / 2n.

[0031] 4. Data Statistics: Transcriptome data from Chinese sea buckthorn leaves were analyzed using Excel software to obtain the distribution and sequence characteristics of SSR loci. The average distance between SSR loci is the ratio of microsatellite length to total length; the frequency of SSR loci is the ratio of the number of microsatellites to the total number of sequences; and G+C stands for guanine and cytosine.

[0032] III. Experimental Results and Analysis 1. Frequency and Average Distance of SSR Loci in the Transcriptome of *Hippophae rhamnoides* in China: The search results for SSR loci are shown in Table 1. Through assembly of the *Hippophae rhamnoides* transcriptome, a total of 68,073 de-redundant unigene sequences were obtained, with a total length of 182,140,905 bp; among them, G+C sequences accounted for 43.40%. As shown in Table 1, a total of 16,684 SSR loci were found, with a total length of 162,719 bp. The frequency of SSR loci occurrence was 11.26%. Of these, 7,664 unigene sequences contained more than one SSR locus, and 1,039 unigene sequences contained SSR loci in complex form. The transcriptome data shows that there is one SSR locus in a sequence with an average length of 10,917.10 bp, meaning there is one SSR locus in every 10.92 kb of sequence length.

[0033] Table 1: Distribution characteristics of SSR loci in the transcriptome of Hippophae rhamnoides in China 2. Basic Characteristics Analysis of SSR Sequences in the Chinese Seabuckthorn Transcriptome: The Chinese seabuckthorn transcriptome possesses a rich variety of SSR motifs. A search was conducted on each SSR repeat motif, and the statistical results are shown in Table 2. Table 2 shows that dinucleotide motifs have the highest repeat frequency, accounting for 50.25% of all SSR sites; followed by trinucleotide motifs, accounting for 44.28%; other motif types, such as tetranucleotides, pentanucleotides, and hexanucleotides, have relatively low proportions, at 3.29%, 0.69%, and 1.49%, respectively. In terms of the distribution distance of each SSR motif type, pentanucleotides have the longest average distribution distance at 3436.62 kb, meaning that an SSR site of a pentanucleotide can be identified on average every 3436.62 kb. This is followed by hexanucleotides, with an average distribution distance of 1597.73 kb, meaning that an SSR site of a hexanucleotide can be identified on average every 1597.73 kb. The shortest distribution distance is for dinucleotides, with an average distribution distance of 47.30 kb, meaning that an SSR site of a dinucleotide can be identified on average every 47.30 kb. Tetranucleotides and trinucleotides follow in order. Statistical analysis of their occurrence frequency shows that the highest frequency is for dinucleotide motifs, accounting for 5.66%; the lowest frequency is for pentanucleotide motifs, accounting for 0.08%.

[0034] Table 2: Search results of different motif types of SSR loci from Hippophae rhamnoides in China based on transcriptome. 3. Composition and Proportion of SSR Repeat Unit Bases in the Transcriptome of Chinese Seabuckthorn: The composition and proportion of SSR repeat units in the transcriptome of Chinese seabuckthorn are diverse, as shown in Table 3. Table 3 shows that the number of motifs for dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides in the SSR loci of the Chinese seabuckthorn leaf transcriptome are 5, 30, 18, 29, and 76, respectively. The major repeat motif for dinucleotides is AT, with 1158 motifs, accounting for 15.11% of the total SSR loci; the major repeat motif for trinucleotides is GAA, with 306 motifs, accounting for 3.99% of the total SSR loci; the major repeat motif for tetranucleotides is AAAT, with 26 motifs, accounting for 0.34% of the total SSR loci; there are two major repeat motifs for pentanucleotides, AAAAG, with 3 motifs, accounting for 0.04% of the total SSR loci; and the major repeat motif for hexanucleotides is CACTCT, with 3 motifs, accounting for 0.11% of the total SSR loci.

[0035] Table 3: Molecular sequence characteristics of SSR site repetitions in the transcriptome of Hippophae rhamnoides in China 4. Repeat Count of Each Molecular Unit in the Chinese Seabuckthorn Transcriptome: The repeat counts of SSR loci of five mole types in the Chinese seabuckthorn transcriptome data were retrieved and statistically analyzed. The results are shown in Table 4. Table 4 shows that the repeat counts of SSR loci of different mole types are concentrated between 5 and 12. A total of 7134 SSR loci with repeat counts of 5 to 12 were found, accounting for 93.08% of the total number of retrieved SSR loci. Among them, the SSR loci with 6 repeat counts totaled 2022, accounting for the largest proportion (26.38%) of the total number of retrieved SSR loci; followed by those with 5 and 7 repeat counts, accounting for 24.22% (1856) and 15.84% (1214) of the total number of retrieved SSR loci, respectively. Statistical analysis revealed that trinucleotides were the most numerous among the SSR loci with 5 repeat counts (1622). The total number of SSR sites of different primitive types that occurred more than 12 times accounted for 6.92% of the total number of SSR sites retrieved.

[0036] Table 4: Number of SSR sites with different repetition counts for different primitive types 5. SSR Marker Screening: Based on the SSR locus information obtained from transcriptome data analysis, 4,681 pairs of SSR primers were designed using 6,196 SSR loci, with a design success rate of 75.55%. To verify the effectiveness of SSR, 130 pairs of SSR primers of different types were randomly selected for verification. From the successfully amplified primers, 20 pairs of primers with good polymorphism were screened, as shown in Table 5. The screening electrophoresis diagram is shown below. Figure 1 As shown.

[0037] Table 5: SSR Primers 6. SSR Genetic Diversity Analysis: Twenty pairs of SSR primers were used to amplify and detect the genetic diversity of 95 Chinese sea buckthorn resources (Table 6) using fluorescence capillary electrophoresis. Genetic diversity parameters were calculated (Tables 7-9). SSR Hardy-Weinberg equilibrium analysis was performed to determine the deviation values ​​at these 20 loci. For loci SZ01, SZ16, SZ111, and SZ124, P < 0.01 indicated significant deviations and genetic instability. Using these 20 primer pairs, 147 allele loci were detected in the 95 Chinese sea buckthorn lines. The Na values ​​for each locus were calculated using PopGen32, ranging from 4 to 15, with a mean of 7.350. The Ne values ​​of these sea buckthorn strains ranged from 1.451 to 5.414, with a mean of 3.113. The maximum I value was 1.984, the minimum was 0.646, and the mean was 1.267. The Ho and He values ​​ranged from 0.309 (SZ01) to 0.840 (SZ68) and from 0.312 (SZ01) to 0.820 (SZ100), with mean values ​​of 0.591 and 0.624, respectively. The polymorphism information content (PIC) of each primer ranged from 0.286 to 0.792, with a mean of 0.576, with SZ100 having the highest PIC and SZ01 the lowest. The PIC values ​​of the 12 SSR loci were greater than 0.50. These 12 SSR loci exhibited specific amplification bands in different types of Chinese sea buckthorn resources, with good amplification effects and high polymorphism.

[0038] Table 6: Tested Chinese Seabuckthorn Germplasm Resources Table 7: Hardy-Weinberg equilibrium analysis of SSR Note: This indicates that P < 0.05. This indicates that P < 0.01.

[0039] Table 8: Analysis of genetic diversity parameters of SSR primers Table 9: Analysis of genetic diversity parameters of SSR primers 7. Cluster Analysis: Based on 20 pairs of polymorphic markers, the genetic similarity coefficient was calculated using PopGen32 software, and the results were analyzed using MEGA 5.1 software for 100 Chinese sea buckthorn accessions. Hippophae rhamnoides subsp. sinensis Cluster analysis and principal component analysis (PCA) were performed on the resources. The cluster analysis results are as follows: Figure 2As shown, the results indicate that all germplasm can be divided into nine main groups (Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ, Ⅸ; specific material numbers are detailed in Table 6). Group II includes the following samples: 111 Kelan, 112 Zuoyun, 8 Youyu, 1 Pianguan, 36 Jingle, 106 Youyu, 92 Kelan, 91 Kelan, 15 Wuzhai, 114 Wuzhai, 39 Fangshan, and 25 Ningwu. Intra-group analysis showed relatively high genetic similarity between samples 111 Kelan and 112 Zuoyun, and between 91 Kelan and 15 Wuzhai, indicating close kinship. Overall clustering structure showed relatively small genetic distance between groups Ⅷ and Ⅸ, indicating that these two groups are more closely related than the other groups. Based on the results of this study, the genetic relationships of the natural distribution of sea buckthorn in China show the following geographical distribution pattern: along the Yellow River Basin, the genetic distance between populations distributed from south to north shows an increasing trend, that is, the kinship gradually becomes more distant with increasing latitude (from south to north).

[0040] 8. Genetic Structure Analysis: Further genetic structure maps of the Chinese sea buckthorn germplasm resources were constructed using Structure 2.3.4, CLUMPP_Windows.1.1.2, and Distruct 1.1 computational mapping software. The results showed that ΔK had two peaks at K=4 and 7 for 100 germplasm accessions. Figure 3 , Figure 4 , Figure 5 This indicates that the genetic structure of the tested Chinese sea buckthorn germplasm is relatively complex, but there is a large amount of germplasm mixing among populations, and there is mutual gene infiltration.

[0041] The SSR loci in the transcriptome data of *Hippophae rhamnoides* from China exhibit a relatively high frequency and short average distribution distance, indicating a large number and variety of SSR loci and suggesting high polymorphism potential. The frequency of SSR loci generally decreases with increasing repeat number. Therefore, most SSR loci in the sequenced *Hippophae rhamnoides* transcriptome from China have polymorphic potential and low mutation frequency. Factors influencing the content of short repeat units in SSRs include species evolution time and species variation frequency. The *Hippophae rhamnoides* transcriptome shows a rich variety of SSR repeat types and a high content of short repeat units. Besides single nucleotide repeats, dinucleotide repeats are the most common loci in *Hippophae rhamnoides* EST-SSRs, followed by trinucleotide repeats. Regarding the types of SSR repeat motifs in *Hippophae rhamnoides*, motifs with higher AT content appear more frequently, and longer SSR repeat motifs occur less frequently. The rich diversity of SSR loci in the coding regions of the *Hippophae rhamnoides* genome provides possibilities for SSR primer development and subsequent research.

[0042] By assembling the transcriptome of *Hippophae rhamnoides* from China, a total of 68,073 de-redundant Unigene sequences were obtained, with a total length of 182,140,905 bp; among them, G+C sequences accounted for 43.40%. As shown in Table 1, a total of 16,684 SSR loci were found, with a total length of 162,719 bp, and an SSR locus frequency of 11.26%. The relatively high frequency and short average distribution distance of SSR loci in the *Hippophae rhamnoides* transcriptome data from China indicate a large number and variety of SSR loci.

[0043] Studies have shown that SSR loci with high repetition frequency have higher polymorphic potential. A total of 7134 SSR loci with repetition frequency concentrated between 5 and 12 times across five motif types were identified, accounting for 93.08% of the total retrieved SSR loci. Repetition types with more than 12 repetitions accounted for 6.92% of the total retrieved SSR loci. Based on these results, it can be concluded that *Hippophae rhamnoides* (Chinese sea buckthorn) has high polymorphic potential. The frequency of SSR loci generally decreases with increasing repetition frequency. Therefore, most SSR loci in the sequenced *Hippophae rhamnoides* transcriptome have polymorphic potential and low mutation frequency, making them suitable for the design and development of highly polymorphic *Hippophae rhamnoides* SSR primers.

[0044] Twenty primer pairs developed from the transcriptome of Chinese sea buckthorn for EST-SSR detection identified 147 allelic loci in 95 Chinese sea buckthorn varieties. The Na values ​​for each locus, calculated using PopGen32, ranged from 4 to 15, with a mean of 7.350. The Ne values ​​for these sea buckthorn varieties ranged from 1.451 to 5.414, with a mean of 3.113. The maximum I value was 1.984, the minimum was 0.646, and the mean was 1.267. The mean Ho and He values ​​were 0.591 and 0.624, respectively. The polymorphism information content (PIC) of each primer ranged from 0.286 to 0.792, with a mean of 0.576.

[0045] Chinese sea buckthorn is a primitive group of the genus Hippophae rhamnoides, a subspecies found only in my country. Its distribution is closely related to light, heat, water, and soil conditions. It is found in areas with an annual precipitation of 400–700 mm and an annual total solar radiation of 130–140 kcal / cm². 2The number of days with temperatures ≥5℃ is 200-225 days per year, and the soil is relatively loose, gravelly, or sandy. The area where isopleths of various ecological factors intersect in the middle reaches of the Yellow River in Shanxi Province, and the large area of ​​natural Hippophae rhamnoides forests in Shanxi Province, support the view that the middle reaches of the Yellow River are the origin of Hippophae rhamnoides in China, and may also be a center of taxonomic differentiation, a center of primitive taxa, and a center of origin for Hippophae rhamnoides. Overall, the SSR loci in the transcriptome of Hippophae rhamnoides in China have a high frequency of occurrence, are closely distributed, and exhibit rich repetition types and repetition motifs. Primers developed based on these characteristics have the potential for polymorphism. However, the exploration of SSR loci in Hippophae rhamnoides in China remains at the level of feature analysis. Further large-scale development and widespread application of SSR primers, analysis of genetic diversity among Hippophae rhamnoides varieties in China, and construction of genetic maps require relevant experimental verification on more varieties.

[0046] In practice, the strains of the Chinese sea buckthorn subspecies are often simply referred to as "Chinese sea buckthorn," lacking detailed strain differentiation, which poses certain difficulties for the identification and evaluation of Chinese sea buckthorn germplasm resources. Germplasm materials (cultivated germplasm, semi-cultivated germplasm, and wild germplasm) are the foundation of forest tree breeding work, and the identification of genetic diversity is essential for the preservation of germplasm genetic resources and the prevention of loss. Analyzing germplasm genotypes using RNA-Seq SSR markers can provide useful information for sea buckthorn breeding projects, such as effective selection of hybrid parents and the development of association markers for important quality traits of sea buckthorn, thus providing a scientific basis for the in-depth development and utilization of the economic value of sea buckthorn.

[0047] The advantages of SSRs in Chinese sea buckthorn are ease of operation, reproducibility, high accuracy, rich variation, good interspecific transmissibility, and a low probability of invalid alleles. Furthermore, because SSRs are identified in coding regions of the genome, they can be applied to fields such as Chinese sea buckthorn strain identification, genetic diversity research, and core germplasm evaluation. The DNA molecular identity card of germplasm resources is closely related to the geographical origin of the material; similar geographical origins result in similar molecular identity cards, while significant differences in geographical origin make molecular identity cards easier to identify.

[0048] This invention utilizes transcriptome mining to extract a large number of SSR sequences, and combines SSR capillary electrophoresis technology to screen out the top 20 SSR loci with high Na, I, and PIC values ​​(measures of genetic differences), as detailed in Table 5. This information is used to create molecular identity information for Chinese sea buckthorn and apply it to the identification of the geographical origin of Chinese sea buckthorn.

[0049] The amplification results are encoded using numbers, letters, and symbols. The arrangement and combination of 20 primer marker pairs are used to generate barcodes for 100 core germplasms. For example, the code for germplasm x005 is 7F3S23332SLC1K7YSCDN. The first digit 7 indicates that the amplified fragment of primer STR1 in germplasm x005 is 274 / 274; the second digit F indicates that the amplified fragment of STR102 in germplasm x005 is 222 / 234; and the third digit 3 indicates that the amplified fragment in germplasm x005 is 211 / 211. This is the code for germplasm x005. Similarly, other codes represent corresponding information based on the same definition. See Table 6 for the specific codes for the 100 germplasms. The germplasm codes are then imported into barcode generation software to generate 100 barcodes. The barcode codes for the 100 germplasms are shown in Table 10.

[0050] Table 10: Germplasm Resource Coding + Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Polymorphic primers for SSR molecular markers in the Chinese sea buckthorn genome, characterized by: The primers consist of the following 20 pairs of polymorphic primers for SSR molecular markers, and their nucleotide sequences are as follows: The forward primer for SZ01 is shown in SEQ ID NO. 1; the reverse primer is shown in SEQ ID NO.

2. The forward primer for SZ03 is shown in SEQ ID NO. 3; the reverse primer is shown in SEQ ID NO.

4. The forward primer for SZ08 is shown in SEQ ID NO. 5; the reverse primer is shown in SEQ ID NO.

6. The forward primer for SZ16 is shown in SEQ ID NO. 7; the reverse primer is shown in SEQ ID NO.

8. The forward primer for SZ34 is shown in SEQ ID NO. 9; the reverse primer is shown in SEQ ID NO.

10. The forward primer for SZ56 is shown in SEQ ID NO. 11; the reverse primer is shown in SEQ ID NO.

12. The forward primer for SZ57 is shown in SEQ ID NO. 13; the reverse primer is shown in SEQ ID NO.

14. The forward primer for SZ67 is shown in SEQ ID NO. 15; the reverse primer is shown in SEQ ID NO.

16. The forward primer for SZ68 is shown in SEQ ID NO. 17; the reverse primer is shown in SEQ ID NO.

18. The forward primer for SZ79 is shown in SEQ ID NO. 19; the reverse primer is shown in SEQ ID NO.

20. The forward primer for SZ88 is shown in SEQ ID NO. 21; the reverse primer is shown in SEQ ID NO.

22. The forward primer for SZ95 is shown in SEQ ID NO. 23; the reverse primer is shown in SEQ ID NO.

24. The forward primer for SZ97 is shown in SEQ ID NO. 25; the reverse primer is shown in SEQ ID NO.

26. The forward primer for SZ100 is shown in SEQ ID NO. 27; the reverse primer is shown in SEQ ID NO.

28. The forward primer for SZ102 is shown in SEQ ID NO. 29; the reverse primer is shown in SEQ ID NO.

30. The forward primer for SZ110 is shown in SEQ ID NO. 31; the reverse primer is shown in SEQ ID NO.

32. The forward primer for SZ111 is shown in SEQ ID NO. 33; the reverse primer is shown in SEQ ID NO.

34. The forward primer for SZ114 is shown in SEQ ID NO. 35; the reverse primer is shown in SEQ ID NO.

36. The forward primer for SZ124 is shown in SEQ ID NO. 37; the reverse primer is shown in SEQ ID NO.

38. The forward primer for SZ126 is shown in SEQ ID NO. 39; the reverse primer is shown in SEQ ID NO.

40.

2. The polymorphic primers for the SSR molecular marker of the Chinese sea buckthorn genome according to claim 1, characterized in that: The 5' end of the forward primer is modified with M13, the sequence of which is 5'-TGTAAAACGACGGCCAGT-3'; a fluorescent group is attached to the 3' end of the synthesized primer.

3. The polymorphic primers for the SSR molecular marker of the Chinese sea buckthorn genome according to claim 2, characterized in that: The fluorescent group is one of FAM, HEX, ROX, and NED.

4. The application of the polymorphic primers of the SSR molecular markers of the Chinese sea buckthorn genome as described in any of claims 1-3 in the analysis of the genetic structure and genetic diversity of Chinese sea buckthorn populations.

5. The application of the polymorphic primers of the Chinese sea buckthorn genome SSR molecular markers according to any one of claims 1-3 in the molecular marker-assisted breeding of Chinese sea buckthorn.

6. The polymorphic primers of the SSR molecular markers of the Chinese sea buckthorn genome as described in any one of claims 1-3 are used as molecular identity information of Chinese sea buckthorn in the identification of the geographical origin of Chinese sea buckthorn.

7. The application according to claim 1, characterized in that: Includes the following steps: 1) Extract genomic DNA from Chinese sea buckthorn; 2) The genomic DNA extracted in step 1) was amplified by PCR using the primers shown in SEQ ID NO. 1-SEQ ID NO. 40 to obtain the amplification products; 3) Perform polyacrylamide gel electrophoresis and / or capillary electrophoresis on the amplification products obtained in step 2). 4) Data analysis of SSR locus polymorphism in the nuclear genome to conduct genetic structure and genetic diversity analysis of Chinese sea buckthorn populations and molecular marker-assisted breeding.

8. The application according to claim 7, characterized in that: The PCR amplification system consisted of: 17.5 μL of 2×Taq PCR premixed reagent II, 10 μmol·L⁻¹ -1 1.0 μL each of forward and reverse primers, 10 μmol·L⁻¹ -1 0.5 μL of M13 fluorescently labeled primers, 0.8 μL of genomic DNA, and 4.2 μL of ddH2O. The total reaction volume is 25 μL.

9. The application according to claim 7, characterized in that, The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, decreasing by 1℃ per cycle, extension at 72℃ for 30 s, for a total of 6 cycles; denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 30 s, for a total of 30 cycles; extension at 72℃ for 10 min, and storage at 4℃.

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