Development and application of a sex chromosome-specific molecular marker in Hippophae rhamnoides L.

Abstract

The present invention discloses the development and application of a sex chromosome-specific molecular marker of Tibetan seabuckthorn. By using a self-written python script, the sites related to sex specificity are more efficiently identified. According to the existing sites, the second-generation sequencing data of male and female individuals are compared to the female reference genome by IGV software to visually check the reliability of the selected sites. It is found that about 70% of the sites have obvious length polymorphism variation in male and female individuals, which verifies the reliability and accuracy of the method. After the selected sites are subjected to specific primer design, two pairs of primers Hiti_08 and Hiti_17 are successfully designed. The two pairs of sex-specific primers can be applied to the breeding and cultivation of Tibetan seabuckthorn, reasonably allocate the ratio of male and female plants, distinguish the female and male individuals of Tibetan seabuckthorn before flowering, and are not limited by the growth period of Tibetan seabuckthorn plants. A stable and efficient method for identifying the sex of Tibetan seabuckthorn is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant sex chromosome molecular marker primers, and in particular relates to the development and application of a sex chromosome-specific molecular marker primer of Tibetan seabuckthorn. Background Art

[0002] Tibetan sea buckthorn (Hippophae tibetana), belonging to the genus Hippophae of the family Elaeagnaceae, is a dioecious shrub that is unique to the Qinghai-Tibet Plateau. As a plant with significant economic and ecological value, the genus Hippophae has attracted extensive attention from scientific research and industry. Its unique environmental adaptability and rich nutritional ingredients and bioactive compounds make Tibetan sea buckthorn show great application potential in many fields such as anti-aging cosmetics, drug development, health drinks and food processing. In particular, the fruit of Tibetan sea buckthorn has been widely studied and applied because it is rich in vitamin C, flavonoids, essential fatty acids and a variety of compounds with antioxidant, anti-inflammatory and cardiovascular protective effects. However, due to its dioecious characteristics, the inflorescence of Tibetan sea buckthorn is small and the flowering period is short. Even if it blooms, it is not easy to quickly identify it, which brings challenges to the breeding and cultivation of Tibetan sea buckthorn.

[0003] In the commercial cultivation of seabuckthorn, it is crucial to properly balance the ratio of male and female plants. For Tibetan seabuckthorn seedlings, the sex can only be identified and the excess male plants can be removed after they bloom for 3-4 years. Relying on sex identification after flowering not only increases the waste of resources, but also increases the difficulty of management. Ali and Kaul (2012) found that the number of male plants in most populations was greater than that of female plants in a survey of the abundance of female and male plants in natural populations of Tibetan seabuckthorn. Therefore, early and accurate sex identification is particularly important, which can effectively reduce maintenance costs and improve economic benefits. In recent years, molecular marker technology has been widely used for sex identification in a variety of dioecious plants, such as Actinidia arguta, spinach (Spinacia oleracea), papaya (Caricapapaya), etc. However, the current research on sex-specific molecular markers for the genus Seabuckthorn is still very limited.

[0004] At present, the research on sex identification of Hippophae rhamnoides mainly focuses on other species of Hippophae rhamnoides. For example, Korekar et al. (2010) developed RAPD primers that can specifically amplify female samples of Hippophae rhamnoides, and developed SCAR markers (HrX1 and HrX2) based on this. Chawla et al. (2012) pointed out that the HrX1 marker can accurately distinguish between males and females in Tibetan Hippophae rhamnoides collected from India. However, these markers were not universal in the Tibetan Hippophae rhamnoides species collected in Tibet after preliminary testing, and could not distinguish between males and females. This shows that the applicability of existing Hippophae rhamnoides sex-specific molecular markers in different geographical populations and species varies significantly.

[0005] In recent years, with the rapid development of sequencing technology and the assembly of a large number of high-quality reference genomes of non-model species, new opportunities have been provided for the development of molecular markers for sex in dioecious plants. At present, many studies have successfully designed molecular markers that can distinguish the sex of plants and animals by combining resequencing data with different analysis methods. For example, She et al. (2021) analyzed the resequencing data of 10 spinach (Spinacia oleracea) individuals (5 females and 5 males), identified potential male-specific regions (MSRs) and designed sex-specific primers; Guo et al. (2023) sequenced 15 female and male soft-fruited kiwifruit (Actinidiaarguta) in mixed pools, assembled and designed sex-linked molecular markers based on male-specific kmers; Hui et al. (2022) resequenced 15 female and 15 male long-nosed catfish (Leiocassis longirostris) respectively, identified sex-related regions based on sex-specific single nucleotide polymorphisms (SNPs), and developed molecular markers that accurately distinguish sex.

[0006] Although some studies have attempted to develop molecular markers for sex identification of Tibetan seabuckthorn, these markers are often limited to specific populations or varieties and lack wide applicability. For example, the SCAR marker developed by Korekar et al. (2010) has different application effects in different Tibetan seabuckthorn species, and some markers cannot be specifically amplified in Tibetan seabuckthorn. In addition, most existing sex identification methods rely on complex genomic data and high-cost experimental equipment, which limits their widespread application in actual production. These problems have brought many challenges to the research and application of sex identification of Tibetan seabuckthorn. At the same time, the existing technology closest to this study is relatively complex in data analysis, which undoubtedly increases the difficulty of designing specific molecular markers.

[0007] Specific problems with existing technologies include: 1. Limitations of existing sex identification methods. Existing sex identification methods mainly rely on morphological characteristics, which require waiting for the plant to bloom, with a long cycle and low efficiency; 2. Applicability issues of existing molecular marker technologies. Current molecular marker technologies are mostly limited to specific populations or varieties, lack versatility, and cannot meet the sex identification needs of Tibetan sea buckthorn; 3. Lack of efficient sex identification technology: There is currently a lack of a fast, accurate, and cost-effective sex identification technology, which has hindered the research and commercial application of Tibetan sea buckthorn; 4. The recent methods for developing male-female specific molecular markers are relatively complex.

[0008] Therefore, in view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention aims to develop sex-specific molecular markers suitable for Tibetan seabuckthorn through a more efficient method, and provide a method for identifying the sex of seabuckthorn using the molecular markers to solve the problem of its sex identification. Summary of the invention

[0009] In view of this, the first object of the present invention is to provide a sex chromosome-specific molecular marker primer combination for identifying the sex of Tibetan seabuckthorn, the nucleotide sequences of which are shown in Hiti_08 and Hiti_17:

[0010] The primer sequence shown in Hiti_08 comprises: the primer sequence shown in Hiti_08-F and the primer sequence shown in Hiti_08-R;

[0011] Hiti_08-F: 5′-TTAGGTCACCTCCTCATGTGTTTAAT-3′;

[0012] Hiti_08-R: 5′-GACTAAAACACCCTTATCAAACGGT-3′;

[0013] The primer sequence shown in Hiti_17 includes: the primer sequence shown in Hiti_17-F and the primer sequence shown in Hiti_17-R;

[0014] Hiti_17-F: 5′-GCACATCTTCCACAGATACATCATC-3′;

[0015] Hiti_17-R: 5′-GGCTAAAGAATTGTCTACATGACCA-3′.

[0016] Another object of the present invention is to disclose a method for identifying the sex of Tibetan seabuckthorn, comprising the following steps:

[0017] S1: Sample collection and DNA extraction: Collect the molecular materials of the Tibetan seabuckthorn to be tested and extract the DNA samples to be tested;

[0018] S2: On the DNA sample to be tested extracted in S1, a conventional PCR amplification experiment was performed using the primer combination shown in Hiti_08 and Hiti_17;

[0019] S3: Separate the PCR products in S2 by 2% agarose gel electrophoresis and send them to a biological company for sequencing; according to the obtained target sequence, the sample with two bands is a male sample, and the sample with one band is a female sample;

[0020] Furthermore, the molecular material in S1 is obtained by sampling fresh leaves of female and male plants of the same number from several existing distribution recording points; the molecular material is dried using silica gel and stored at -20 degrees Celsius; the method for extracting total DNA from the molecular material is the CetyltrimethylammoniumBromide (CTAB) method;

[0021] Furthermore, the PCR reaction system in S2 is as follows: the CR reaction uses a total volume of 30 μL, including 15 μL of TaqMix, 1 μL of DNA template, 1 μL of each forward and reverse primer, and 12 μL of deionized water;

[0022] Furthermore, the PCR reaction program in S2 is: pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, for a total of 35 cycles, and finally full extension at 72°C for 5 minutes, and stored at 4°C;

[0023] On the other hand, another object of the present invention is to provide a method for designing molecular marker primers for identifying the male and female of Tibetan seabuckthorn, comprising the following steps:

[0024] S1: Sample collection and DNA extraction: Collect the molecular materials of the Tibetan seabuckthorn to be tested and extract the DNA samples to be tested;

[0025] S2: Whole genome resequencing and data processing: Screen the DNA samples that meet the quality standards obtained in S1 and send them to a biological company for library construction and sequencing. Use population genetic data analysis methods to detect population variation and screen for male-specific fragments.

[0026] S3: Screening and verification of sex-determining regions: Use python scripts to screen female-specific fragments, and further verify the visual positioning of the screened molecular markers using IGV v.2.16 software; select sequences with long fragment insertions and deletions in males as candidate regions for subsequent primer design;

[0027] S4: Primer design and validation: Synthesize primers for regions linked to sex and conduct preliminary screening in male and female individuals;

[0028] S5: Verification of the universality of primers: Conduct universal tests on more populations for the sex-specific markers initially screened out in S4.

[0029] Furthermore, the molecular material in S1 is obtained by sampling fresh leaves of female and male plants of the same number from several existing distribution record points. The molecular material is dried using silica gel and stored at -20 degrees Celsius; the method for extracting total DNA from the molecular material is the CTAB method;

[0030] Furthermore, the screening operation in S2 is: using 1.0% agarose gel electrophoresis and NanoDrop2000 to evaluate the quality and concentration of the DNA samples, and sending the DNA samples that meet the sequencing quality requirements to a biological company for library construction and sequencing;

[0031] Furthermore, the sequencing described in S2 was performed on a Novasek X Plus PE150 platform with a double-end sequencing depth of 30×; the raw sequencing data was quality controlled and cleaned using Fastp v0.21, and then the data quality was assessed using FastQCv0.11.9; INDELs were identified and filtered in the Genome Analysis Toolkit (GATK4); plinkv.1.9 was used to further filter the data to exclude sites with a minor allele frequency (MAF) < 0.01 and more than 50% missing genotype data; the identification and filtering steps included: removing PCR-generated duplicate sequences, single sample variation detection, population variation detection, and marking INDELs and filtering low-quality sites; the filtering criteria were set as QD<2.0||FS>200.0||SOR>10.0||MQRankSum<-12.5||ReadPosRankSum<-8.0";

[0032] Furthermore, the specific operation of S3 is as follows: using Python scripts, screening for variant sites with long insertion / deletion in male samples; using IGV v.2.16 software to manually check the screened sites, and selecting sequences with long insertion / deletion in males as candidate regions for subsequent primer design;

[0033] Furthermore, in S4, seqkit v2.6.1 software was used to extract the corresponding sequence information from the reference genome. Primers with a length of 200-300 bp were designed in batches using primer3; the designed primers were then compared with the reference genome using blastnv2.5.0 to verify their specificity; primers with high specificity were selected from the designed primers and synthesized by a biological company;

[0034] Further, in S5, the operation of the preliminary selection that has been further universally verified is: a conventional PCR amplification experiment is performed on the batch-synthesized primers on the Tibetan seabuckthorn DNA samples (including 2 female and 2 male individuals) to perform preliminary screening of the ability to distinguish between males and females. The PCR reaction system is a total volume of 30 μL, including 15 μL of TaqMix, 1 μL of DNA template, 1 μL of each forward and reverse primer, and 12 μL of deionized water; the PCR program is: 95°C pre-denaturation for 3 minutes, followed by 95°C denaturation for 15 seconds, 58°C annealing for 15 seconds, 72°C extension for 30 seconds, a total of 35 cycles, and finally 72°C full extension for 5 minutes, and stored at 4°C; the PCR products are separated by 2% agarose gel electrophoresis, and the primers that can effectively distinguish between male and female samples are selected by product length; the screened primers are then used to universally verify more Tibetan seabuckthorn samples that have been distinguished between males and females from different regions.

[0035] Beneficial effects of the present invention:

[0036] By using the python script provided by this project, sex-specific loci can be identified more efficiently. Based on the existing loci, we used IGV software to visualize the comparison of the male and female second-generation sequencing data to the reference genome to verify the reliability of the selected loci, and found that about 70% of the loci had obvious length polymorphism in male and female individuals. This also fully verified the reliability and accuracy of this method.

[0037] This study combined the fragments with significant differences between female individuals and successfully designed two pairs of sex-specific molecular markers for distinguishing between male and female individuals of Tibetan seabuckthorn. After verification on individuals from different groups that have been distinguished between males and females, the results showed that 100% of male and female individuals can be distinguished.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The molecular marker disclosed in the present invention can be applied to the breeding and cultivation of seabuckthorn, reasonably allocate the ratio of male and female plants, distinguish the female and male seabuckthorn individuals before flowering, and is not limited by the growth period of the seabuckthorn plant, providing a stable and efficient method for distinguishing the sex of seabuckthorn.

[0040] The identification method disclosed in the present invention is not limited to specific populations or varieties, has wide applicability, does not rely on complex genetic data and high-cost implementation equipment, and has broader application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0042] Figure 1 In Example 1 of the present invention Figure 1 The two primer pairs Hiti_8 and Hiti_17 were used to verify the sex differentiation of individuals from different regions; DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] (1) Sample collection and DNA extraction

[0046] Male and female samples of Hippophae rhamnoides L. were collected from six different geographical populations (collection sites are shown in Table 1). Four female and four male plants were randomly selected from each population for fresh leaf sampling. The sampled molecular material was dried using silica gel and stored at -20°C. Total DNA was extracted from Hippophae rhamnoides L. leaves using the CTAB method, and the quality and concentration of DNA were assessed using 1.0% agarose gel electrophoresis and NanoDrop 2000 to ensure that the samples met the sequencing quality requirements.

[0047] Table 1. Collection points of Tibetan sea buckthorn samples

[0048]

[0049] (2) Whole genome resequencing and data processing

[0050] DNA samples that met the quality standards were sent to Beijing Novogene Technology Co., Ltd. for DNA library construction and paired-end sequencing was performed on the Novasek X Plus PE150 platform with a sequencing depth of 30×. The raw sequencing data were quality controlled and cleaned using Fastp v0.21, and then the data quality was assessed by FastQC v0.11.9 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). The cleaned data were aligned to the Tibetan Hippophae rhamnoides reference genome using BWAv2.2.1 and then converted to BAM format using samtools v1.19. The identification and filtering of INDELs were performed in the GenomeAnalysis Toolkit (GATK4), and the main steps included the removal of PCR-generated duplicate sequences, single sample variation detection, population variation detection, and the marking of INDELs and the filtering of low-quality sites. The filtering criteria were set as "QD<2.0||FS>200.0||SOR>10.0||MQRankSum<-12.5||ReadPosRankSum<-8.0". The data were further filtered using plinkv.1.9 to exclude sites with minor allele frequency (MAF) <0.01 and missing genotype data of more than 50%. Finally, 6554748 sites were screened out of 7706351 variant sites for further analysis.

[0051] (3) Screening and verification of sex-determining regions

[0052] Considering that the reference genome comes from female plants, the focus is on the alignment of the sequencing data of female and male plants to the same site on the reference genome. Through a self-written Python script, the variant sites with long insertions / deletions in male samples were screened. Finally, the IGV v.2.16 software was used to manually check the screened sites, and the sequences with long insertions / deletions in males were selected as candidate regions for subsequent primer design.

[0053] (4) Primer design and verification

[0054] Based on the variant sites that may be used for primer design, the corresponding sequence information was extracted from the reference genome using seqkitv2.6.1 software. In view of the fact that the length of the variant segments that can be used for primer design was mostly in the range of 30-60bp during manual detection, in order to facilitate the intuitive distinction of male and female samples by product length, primers with an expected length of 200-300bp were designed in batches using primer3. When designing primers, problems such as mismatches, dimers, hairpin structures, and primer dimers were avoided as much as possible. The designed primers were then compared with the reference genome using blastn v2.5.0 to verify their specificity. Through this screening step, primers with high specificity were selected and commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0055] (5) Verification of the universality of primers

[0056] To verify the specificity of the primers, a conventional PCR amplification experiment was first performed on a small amount of Tibetan seabuckthorn DNA samples (including 2 female and 2 male individuals) to preliminarily screen primers that can distinguish between males and females. The PCR reaction used a total volume of 30 μL, including 15 μL of Taq Mix, 1 μL of DNA template, 1 μL of each forward and reverse primer, and 12 μL of deionized water. The PCR program was set as 95℃ pre-denaturation for 3 minutes, followed by 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, and 72℃ extension for 30 seconds, for a total of 35 cycles, and finally 72℃ full extension for 5 minutes, and stored at 4℃. The PCR products were separated by 2% agarose gel electrophoresis, and the primers that can effectively distinguish between male and female samples were selected by product length. The screened primers were then used to verify the accuracy of 48 samples (24 females and 24 males) collected from 6 Tibetan seabuckthorn populations in Tingri, Basu, Dangxiong and Mozhugongka in Tibet. The results showed that both primer pairs could accurately distinguish the female and male samples involved in the test: two bands were produced in all male samples, while one band was produced in female samples (see attached). Figure 1 ).

[0057] Through the above analysis, this study successfully designed two pairs of sex-specific molecular markers for distinguishing male and female plants of Tibetan seabuckthorn, which can be used to accurately identify the sex of Tibetan seabuckthorn (the specific information of the two pairs of primers is shown in Table 2). At the same time, it also further verified the reliability of our new method for selecting male-female specific fragments, which can also be applied to other species.

[0058] Table 2 Sequence information of two pairs of primers Hiti_08 and Hiti_17

[0059]

[0060] Example 2

[0061] A sex chromosome-specific molecular marker primer combination for identifying the sex of Tibetan seabuckthorn, comprising:

[0062] A primer pair of the sequence shown in Hiti_08, comprising: a primer sequence shown in Hiti_08-F and a primer sequence shown in Hiti_08-R;

[0063] A primer pair of the sequence shown in Hiti_17, comprising: a primer sequence shown in Hiti_17-F and a primer sequence shown in Hiti_17-R;

[0064] See Table 2 for details (sequence information of two pairs of primers Hiti_08 and Hiti_17).

[0065] Example 3

[0066] In this embodiment, a method for identifying the sex of Tibetan sea buckthorn comprises the following steps:

[0067] S1: Sample collection and DNA extraction: Collect the molecular materials of the Tibetan seabuckthorn to be tested and extract the DNA samples to be tested;

[0068] S2: On the DNA sample to be tested extracted in S1, a conventional PCR amplification experiment was performed using the primer combination shown in Hiti_08 and Hiti_17;

[0069] S3: Separate the PCR products in S2 by 2% agarose gel electrophoresis and send them to a biological company for sequencing; obtain the target sequence amplified by the primers, and determine the sex of seabuckthorn according to the target sequence. The sample with two bands is a male sample, and the sample with one band is a female sample;

[0070] In this embodiment, the molecular material in S1 is obtained by sampling fresh leaves of female plants and male plants of the same number from several existing distribution recording points; the molecular material is dried using silica gel and stored at -20 degrees Celsius; the method for extracting total DNA from the molecular material is the CTAB method;

[0071] In this embodiment, the PCR reaction system in S2 is as follows: the CR reaction uses a total volume of 30 μL, including 15 μL of TaqMix, 1 μL of DNA template, 1 μL of each forward and reverse primer, and 12 μL of deionized water;

[0072] In this embodiment, the PCR reaction program in S2 is: pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, for a total of 35 cycles, and finally full extension at 72°C for 5 minutes, and stored at 4°C;

[0073] According to the implementation of this embodiment, a method for identifying the sex of Tibetan sea buckthorn is obtained. The method adopts a sex chromosome-specific molecular marker primer combination for identifying the sex of Tibetan sea buckthorn in the technical solution of the present invention. The identification method in the technical solution is compared with the following prior art:

[0074] Prior art 1

[0075] The existing method for sex identification of Tibetan sea buckthorn mainly relies on morphological characteristics, specifically by observing the morphological characteristics of the plant after flowering to distinguish between male and female plants. This method requires waiting for the plant to bloom, which usually takes 3-4 years, to accurately identify the sex of the plant. Its shortcomings are: (1) Long cycle: It is necessary to wait for the Tibetan sea buckthorn to bloom, which usually takes 3-4 years, which greatly prolongs the breeding cycle.

[0076] (2) Low efficiency: Sex identification cannot be performed before flowering, which makes early management difficult and causes serious waste of resources.

[0077] (3) Limited accuracy: Morphological identification methods rely on the external appearance of plants and are easily affected by environmental factors, resulting in insufficient accuracy.

[0078] Compared with the above-mentioned deficiencies of the prior art, the present invention has the following beneficial effects: (1) Short cycle: The present invention utilizes a sex chromosome-specific molecular marker primer combination to perform sex identification at the seedling stage, thus saving the time of waiting for flowering.

[0079] (2) High efficiency: PCR technology can be used to quickly and accurately identify sex, thereby improving breeding and management efficiency.

[0080] (3) High accuracy: The identification method based on molecular markers is not affected by environmental factors and has higher accuracy and reliability.

[0081] Prior art 2:

[0082] Technical content overview: Korekar et al. (2010) developed RAPD primers that can specifically amplify female samples of Hippophae rhamnoides, and based on this, developed SCAR markers (HrX1 and HrX2). These markers can achieve sex identification in specific populations. Its shortcomings:

[0083] (1) Population limitation: The applicability of these SCAR markers varies greatly in different geographical populations, especially in Tibetan sea buckthorn, where specific amplification cannot be achieved.

[0084] (2) Poor applicability: The existing SCAR marker is not universal in Tibetan seabuckthorn and cannot effectively distinguish between males and females.

[0085] (3) High complexity: Reliance on complex genomic data and relatively high-cost experimental equipment limits its widespread application in actual production.

[0086] Compared with the prior art 2, the beneficial effects of the present invention are:

[0087] (1) Wide applicability: The sex-specific molecular markers developed in the present invention have good applicability in different geographical populations, especially showing high accuracy in Tibetan seabuckthorn.

[0088] (2) Strong versatility: Compared with the existing SCAR markers, the molecular markers of the present invention can achieve specific amplification and sex differentiation in multiple Tibetan seabuckthorn species.

[0089] (3) Simplicity: The sex identification method of the present invention is low-cost, has a simple experimental process, and is suitable for large-scale production and commercial application.

[0090] Prior Art 3

[0091] Technical content overview: Chawla et al. (2012) pointed out that HrX1 markers can accurately distinguish males and females in Tibetan sea buckthorn collected from India. However, these markers are not universal in Tibetan sea buckthorn populations in Tibet. Its shortcomings:

[0092] (1) Regional restrictions: HrX1 marker is only effective in Tibetan sea buckthorn populations in a specific geographical area, and is not very effective in Tibetan sea buckthorn populations in the Tibetan region. (2) Poor stability: The amplification results of the marker are unstable and there are significant differences in different samples, which affects its wide application. (3) Complex operation: The experimental process is complicated and time-consuming, making it difficult to promote and apply in actual production.

[0093] Compared with the prior art 3, the beneficial effects of the present invention are:

[0094] (1) Strong regional adaptability: The sex chromosome-specific molecular markers developed by the present invention show high versatility and accuracy in the Tibetan seabuckthorn population in the Tibet region.

[0095] (2) High stability: The amplification results of the marker are stable and highly reproducible, and consistent identification results can be obtained in different samples.

[0096] (3) Simplified operation: The experimental process of the present invention is simple and quick, suitable for the needs of gender identification in actual production, and has high promotion value;

[0097] Through the above comparison, the present invention shows significant advantages in the technology of sex identification of Tibetan sea buckthorn, provides an efficient, accurate and cost-effective method for sex chromosome-specific molecular marker identification, overcomes the shortcomings of the prior art, and has broad application prospects.

[0098] Example 4

[0099] In this embodiment, a method for designing molecular marker primers for distinguishing the sex of Tibetan seabuckthorn is provided, comprising the following steps:

[0100] S1: Sample collection and DNA extraction: Collect the molecular materials of the Tibetan seabuckthorn to be tested and extract the DNA samples to be tested;

[0101] S2: Whole genome resequencing and data processing: Screen the DNA samples that meet the quality standards obtained in S1 and send them to a biological company for library construction and sequencing. Use population genetic data analysis methods to detect population variation and screen for male-female specific fragments.

[0102] S3: Screening and verification of sex-determining regions: Python scripts were used to screen male-specific fragments, and the selected molecular markers were further verified by visual positioning using IGV v.2.16 software; sequences with long fragment insertions and deletions in males were selected as candidate regions for subsequent primer design;

[0103] S4: Primer design and validation: Synthesize primers for regions linked to sex and conduct preliminary screening in male and female individuals;

[0104] S5: Verification of the universality of primers: Conduct universal tests on more populations for the sex-specific markers initially screened out in S4.

[0105] In this embodiment, the molecular material in S1 is selected from several existing distribution record points, and the same number of female plants and male plants are selected for fresh leaf sampling. The molecular material is dried using silica gel and stored at -20 degrees Celsius; the method for extracting total DNA from the molecular material is the CTAB method; regarding the distribution points, the distribution points selected in this embodiment are the distribution points shown in Table 1, but are not limited to only including the above distribution points.

[0106] In this embodiment, the screening operation in S2 is: using 1.0% agarose gel electrophoresis and NanoDrop2000 to evaluate the quality and concentration of the DNA sample, and selecting the DNA sample that meets the sequencing quality requirements;

[0107] In this embodiment, the sequencing described in S2 is double-end sequencing on the Novasek X Plus PE150 platform, and the sequencing depth is 30×; the raw sequencing data is subjected to quality control and data cleaning using Fastp v0.21, and then the data quality is evaluated by FastQCv0.11.9; INDELs are identified and filtered in the Genome Analysis Toolkit (GATK4); plinkv.1.9 is used to further filter the data to exclude sites with minor allele frequency (MAF) <0.01 and missing genotype data exceeding 50%; the identification and filtering steps include: removing repeated sequences generated by PCR, single sample variation detection, population variation detection, and marking INDELs and filtering low-quality sites; the filtering criteria are set to "QD<2.0||FS>200.0||SOR>10.0||MQRankSum<-12.5||ReadPosRankSum<-8.0";

[0108] In this embodiment, the specific operation of S3 is: using Python script to screen the variant sites with long insertion / deletion in male samples; using IGV software to manually check the screened sites, and select the sequences with long insertion / deletion in males as candidate regions for subsequent primer design;

[0109] In this embodiment, in S4, seqkit v2.6.1 software is used to extract the corresponding sequence information from the reference genome. Primers that are expected to produce products with a length of 200-300 bp are designed in batches using primer3; the designed primers are then compared with the reference genome using blastn v2.5.0 to verify their specificity; primers with high specificity are selected from the designed primers and synthesized by a biological company;

[0110] In this embodiment, in S5, the operation of the universal verification is: a conventional PCR amplification experiment is performed on the batch synthesized primers on the Tibetan seabuckthorn DNA samples (including 2 female and 2 male individuals). The PCR reaction system is a total volume of 30 μL, including 15 μL of Taq Mix, 1 μL of DNA template, 1 μL of each forward and reverse primer, and 12 μL of deionized water; the PCR program is: 95°C pre-denaturation for 3 minutes, followed by 95°C denaturation for 15 seconds, 58°C annealing for 15 seconds, 72°C extension for 30 seconds, a total of 35 cycles, and finally 72°C full extension for 5 minutes, and stored at 4°C; the PCR products are separated by 2% agarose gel electrophoresis, and the primers that can effectively distinguish between male and female samples are selected by product length; the screened primers are used to verify the accuracy of samples from several Tibetan seabuckthorn populations.

[0111] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. According to the content of this specification, many modifications and changes can be made. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A sex chromosome-specific molecular marker primer combination for identifying the sex of Tibetan seabuckthorn, characterized in that: The primer combination consists of Hiti_08 and Hiti_17; The primer sequence of Hiti_08 is as follows: Hiti_08-F:5'-TTAGGTCACCTCCTCATGTGTTTAAT-3'; Hiti_08-R:5'-GACTAAAACACCCTTATCAAACGGT-3'; The primer sequence of Hiti_17 is as follows: Hiti_17-F:5'-GCACATCTTCCACAGATACATCATC-3'; Hiti_17-R:5'-GGCTAAAGAATTGTCTACATGACCA-3'.

2. The use of the primer combination according to claim 1 in identifying the sex of Tibetan seabuckthorn, characterized in that: The primer combination is used for PCR amplification experiment, and the gender is determined according to the number of product bands after the primer combination is amplified. The sample with two bands is a male sample, and the sample with one band is a female sample.

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