SNP marker and identification method for identifying female and male siraitiae fructus plants

Abstract

By means of GWAS data association analysis of multiple years and multiple repetitions of phenotypic data of 125 varieties collected by Monk Fruit Corp. and wild natural varieties collected in the Guilin region, it is found that the sex-determining region is located in the 0-1 mbps range of chromosome 3 of the genome. Therefore, provided is an SNP region for early identification of female and male Siraitiae fructus plants. In this region, the female plant and the male plant have their own haplotypes. Finally, a group of haplotypes of male and female plants are screened out, and any SNP marker in the haplotypes can be used for development of SNP molecular markers and identification of male and female plants.

Description

SNP markers and identification methods for identifying male and female Momordica grosvenori plants Technical Field

[0001] This application relates to the fields of genetic engineering and molecular biology, and is directed to the identification and application of a region that determines the sex of Momordica grosvenori. Background Art

[0002] Momordica grosvenori is a dioecious plant. Its male and female plants cannot be distinguished by the naked eye in terms of morphology. The sex can only be distinguished when they bloom. Although the number of seedlings used in production is relatively small, and most of them are tissue culture propagated seedlings, the planting of seedlings is particularly important in the breeding of new varieties with high yield and high glycoside content. In order to improve the quality of seedlings, protect them from the influence of viruses and diseases, and improve their root systems and weak growth potential, if male and female plants cannot be distinguished during the seedling stage, then planting a large number of female plants that have not been screened for males will waste a lot of manpower and land resources. Therefore, the development of a marker for rapid seedling sex identification before transplanting is of great production significance, especially to promote the hybrid advantage of Momordica grosvenori and improve the efficiency of breeding.

[0003] Previous methods for selecting male and female plants typically involved selectively removing male plants during flowering to reduce plant management requirements. In 2013, Zhang Huixin et al. published a patent titled "A SCAR Molecular Marker for Identifying the Sex of Momordica grosvenori." This marker enabled the use of PCR to screen male and female plants during the early seedling stage. However, the amplified fragments in this patent could not be definitively linked to the male and female genes or the genomic sex-control loci. Furthermore, this marker is not necessarily universally applicable, as it has not been validated in other Momordica grosvenori varieties as published in the patent. Furthermore, this method suffers from slow and low throughput.

[0004] The male-to-female ratio in the offspring of Momordica grosvenori is 1.3:1, with more male plants than females. This has limited the promotion of hybrid seedlings due to their economic value. Due to the lack of a complete reference genome for Momordica grosvenori, its genetic polymorphism cannot be systematically compared and analyzed, and molecular marker screening and verification cannot be performed. At the same time, the differences between male and female plants at the genomic level are difficult to compare due to the lack of a reference genome, making it even more difficult to identify the genes that determine male and female sex. Although the genetic ratio of male and female Momordica grosvenori is known, there is no similar model to explain the genetic mechanism of male-to-female sex in Momordica grosvenori. Furthermore, when locating the male-to-female genes in Momordica grosvenori, a complete reference genome is also required to locate and identify genes linked to the traits.

[0005] In 2018, Xia et al. assembled a Luo Han Guo genome, but this genome was not assembled to the chromosome level, and more than 2,000 contig fragments that could not be assembled remained, making any gene mapping very difficult.

[0006] Summary of the Invention

[0007] Due to the lack of a complete genome and male-female identification markers, the improvement and large-scale screening of dioecious Momordica grosvenori has long been limited by conventional breeding. To address the problems existing in the prior art, this application splices and assembles the whole genome of a commercial variety from Giffords, and compares the male and female plants of multiple different varieties on different chromosomes. The purpose is to provide a haplotype that distinguishes males from females on a chromosome region, as well as SNP molecular markers in this haplotype and their application.

[0008] This application can solve the accuracy and reliability issues of identifying the sex of Momordica grosvenori. This technology can determine the sex of Momordica grosvenori plants at the seedling stage, avoiding the problem of wasting germplasm resources and costs. At the same time, this technology can be applied to different varieties of Momordica grosvenori plants, improving the universality and practicality of research.

[0009] Specifically, this application adopts the following technologies:

[0010] 1. A SNP marker for identifying female and male Momordica grosvenori plants, wherein the SNP marker is a site with a p-value greater than 25.

[0011] 2. The SNP marker according to item 1, wherein the nucleotide sequence of the SNP marker is as shown in SEQ ID NO: 1, and the position is the 122nd position of the nucleotide sequence, wherein the nucleotide represented by S in SEQ ID NO: 1 is G or C.

[0012] 3. The SNP marker according to item 1 or 2, wherein when the alleles at the S position of the Momordica grosvenori diploid are G and C, the Momordica grosvenori is a male plant, and when the alleles at the S position of the Momordica grosvenori diploid are C and C, the Momordica grosvenori is a female plant.

[0013] 4. A primer for detecting the SNP marker described in any one of items 1 to 3, comprising:

[0014] Forward primer 1:

[0015] 5'-GAAGGTGACCAAGTTCATGCTTCAACCATTGGGGATGGAG-3';

[0016] Forward primer 2:

[0017] 5'-GAAGGTCGGAGTCAACGGATTTCAACCATTGGGGATGGAC-3';

[0018] Reverse primer: 5′-GTAGAAGCGGCTGCTTTTACTGG-3′.

[0019] 5. A detection reagent or kit for detecting the SNP marker according to any one of items 1 to 3, comprising the primer according to item 4.

[0020] 6. Use of the SNP marker according to any one of items 1 to 3, the primer according to item 4, or the detection reagent or kit according to item 5 in identifying or screening female and male Momordica grosvenori plants.

[0021] 7. Use of the SNP marker according to any one of items 1 to 3, the primer according to item 4, or the detection reagent or kit according to item 5 in marker-assisted breeding of Momordica grosvenori.

[0022] 8. Use of the SNP marker according to any one of items 1 to 3, the primer according to item 4, or the detection reagent or kit according to item 5 in Momordica grosvenori hybrid breeding.

[0023] 9. A method for distinguishing male and female Momordica grosvenori plants, wherein the SNP marker for distinguishing male and female Momordica grosvenori plants is detected to predict whether the Momordica grosvenori plant is a female plant or a male plant, wherein the SNP marker is the SNP marker described in any one of items 1 to 3.

[0024] 10. The method according to claim 9, comprising:

[0025] Extracting genomic DNA from the tested Momordica grosvenori;

[0026] Using the genomic DNA of the tested Momordica grosvenori as a template, PCR amplification is performed using the primers described in item 4 to obtain a PCR amplification product;

[0027] Detect the PCR amplification product. If the alleles at the S position of the diploid sequence of the amplified product are G and C, the Momordica grosvenori is a male plant; when the alleles at the S position of the diploid sequence of the Momordica grosvenori are C and C, the Momordica grosvenori is a female plant.

[0028] Effects of the Invention

[0029] This application uses 125 varieties collected by Giffords and wild natural varieties collected in the Guilin area to perform GWAS data association analysis through multi-year and multi-repeated phenotypic data, and finds that the region determining sex is located in the 0-1 Mbps interval of chromosome 3 of the genome. Therefore, this application provides a SNP region for early identification of male and female Momordica grosvenori plants. Female and male plants in this region have their own haplotypes. Finally, a group of haplotypes of male and female plants are screened, and any SNP marker in the region can be used for SNP molecular marker development and identification of male and female plants.

[0030] This study directly sequenced 125 DNA samples from different varieties and found the common haplotypes of male and female plants. The haplotypes of male and female plants are not restricted by season, environment, or expression.

[0031] The screening method of the present application is technically efficient, rapid, and accurate. The sex of the plants can be determined with a detection accuracy of 96% without the need to plant them until they bloom, which greatly reduces the cost of planting and increases the efficiency of Momordica grosvenori hybrid breeding.

[0032] The research and application of this application alleviate the technical problem of lacking an accurate and complete reference genome and effective and efficient molecular biological markers in the molecular breeding research of Momordica grosvenori. The application of this technology will help improve the efficiency and quality of Momordica grosvenori breeding and promote the development of the Momordica grosvenori industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are provided to facilitate a better understanding of the present application and do not constitute an undue limitation on the present application.

[0034] Figure 1 is a Manhattan plot of the SNP marker site, with the horizontal axis being the chromosome number and the vertical axis being the association analysis-log P value.

[0035] Figure 2 is a quantile map of SNP sites for male-female trait association analysis. DETAILED DESCRIPTION

[0036] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0037] The present application provides a SNP marker for identifying female and male Momordica grosvenori plants, wherein the SNP marker is a site with a p-value greater than 25.

[0038] The term "single nucleotide polymorphism" or "SNP" refers to a change in the DNA sequence or a genetic variant that occurs when one nucleotide [e.g., adenine (A), thymine (T), cytosine (C), or guanine (G)] is changed to another nucleotide in the genomic sequence.

[0039] The term "p-value" is a parameter used to determine the results of a hypothesis test. It can also be used to compare different distributions using the rejection region of the distribution. It was first proposed by R.A. Fisher.

[0040] The p-value (P-value) is the probability of a more extreme result than the sample observation occurring when the null hypothesis is true. If the P-value is small, the probability of the null hypothesis occurring is very small. However, if it does occur, the principle of small probability is used to justify rejecting the null hypothesis. The smaller the P-value, the stronger the reason for rejecting the null hypothesis. In short, the smaller the P-value, the more significant the result.

[0041] In this application, the p-value refers to the statistical significance indicator of the association between genes or genetic variants and traits in GWAS analysis. A smaller p-value indicates that the observed association result is unlikely to be caused by random factors, which increases the confidence in rejecting the null hypothesis. In this application, the p-value is expressed as (-log 10 (p)) indicates.

[0042] In the present application, each SNP marker site with a p-value greater than 25 is considered to be a SNP associated with the identification of female and male Momordica grosvenori plants and can be used to identify female and male Momordica grosvenori plants.

[0043] Momordica grosvenori is the fruit of a perennial vine in the Cucurbitaceae family. Also known as Lahan fruit, false bitter melon, light fruit turtle, golden incorporeal, Luohanbiao, and naked turtle bark, it is hailed as the "immortal fruit". Its leaves are heart-shaped, and it is dioecious. It blooms in summer and bears fruit in autumn. It is one of the first batch of medicinal and edible materials approved by the country, and its main function is to relieve cough and reduce phlegm. The fruit has high nutritional value and is rich in vitamin C (400 mg to 500 mg per 100 g of fresh fruit) as well as glycosides, fructose, glucose, protein, lipids, etc. There are many varieties of Momordica grosvenori, and they have different names according to different classification methods. The SNP sites involved in this application can identify any variety of Momordica grosvenori without limitation, such as the common long beach fruit, green skin fruit, red hair fruit, tea mountain fruit, Lajiang fruit, winter melon fruit, etc.

[0044] In a specific embodiment, the nucleotide sequence of the SNP marker is as shown in SEQ ID NO: 1, and the position is the 122nd position of the nucleotide sequence, wherein the nucleotide represented by S in SEQ ID NO: 1 is G or C.

[0045] In a specific embodiment, when the alleles at the S position of the Momordica grosvenori diploid are G and C, the Momordica grosvenori is a male plant; when the alleles at the S position of the Momordica grosvenori diploid are C and C, the Momordica grosvenori is a female plant.

[0046] The present application provides a primer set for identifying SNPs in female and male Momordica grosvenori plants, including a forward primer and a reverse primer. The primer set includes a primer pair for detecting the above-mentioned SNP marker site.

[0047] In a specific embodiment, exemplary primer pairs are shown below, wherein the primers include:

[0048] Forward primer 1, as shown in SEQ ID NO: 2,

[0049] 5'-GAAGGTGACCAAGTTCATGCTTCAACCATTGGGGATGGAG-3';

[0050] Forward primer 2, as shown in SEQ ID NO: 3,

[0051] 5'-GAAGGTCGGAGTCAACGGATTTCAACCATTGGGGATGGAC-3';

[0052] Reverse primer, as shown in SEQ ID NO: 4,

[0053] 5'-GTAGAAGCGGCTGCTTTTACTGG-3'.

[0054] For the specific base sequences of the above-mentioned exemplary primer pairs, as long as they can specifically recognize their respective specific recognition regions under the conditions for PCR (preferably, annealing and self-annealing do not occur between primers used in a single reaction vessel), one or more bases can be replaced with other bases, or one or more bases can be added to the 3' or 5' end. Here, the term "multiple" refers to, for example, 2 to 3 bases. When adding one or more bases to a primer, it is preferably added to the 5' end of the primer.

[0055] The identity of the base sequence obtained by replacing one or more bases in the specific base sequence of the primer exemplified above with other bases and the base sequence before substitution (i.e., the base sequence shown in the sequence number) is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more.

[0056] According to the embodiments of the present application, the above primers have very good specificity, and the above primers can be used to accurately and effectively perform PCR amplification on the fragment where the SNP marker site related to the tested Momordica grosvenori is located.

[0057] The present application provides a SNP marker detection reagent or kit as described in any one of the above items, wherein any one of the primers described above.

[0058] In a specific embodiment, the kit further comprises reagents for extracting genomic DNA from a sample and reagents for performing a PCR reaction using the primers.

[0059] In a specific embodiment, the reagent for extracting genomic DNA from a sample can be an existing kit.

[0060] In one embodiment, the reagents for performing a PCR reaction using the primer set can be selected from DNA polymerase, PCR buffer, dNTP mixture, and aqueous medium, or a premix of the above reagents. For example, the premix is ​​selected from EXTaq (HS): Premix Ex Taq™ Hot Start Version, Takara (RR030Q).

[0061] The PCR buffer can generally provide the most suitable conditions for the enzymatic reaction in the PCR system. The buffer only needs to play the above-mentioned role.

[0062] The dNTP mixture is usually used as a raw material in DNA synthesis, and specifically may include dATP, dGTP, dTTP, dCTP, etc.

[0063] The aqueous medium can generally be used to adjust the concentration of each component in the PCR system and can generally be used as a dilution solvent.

[0064] The present application further provides the use of any of the above-described SNP markers, primers, detection reagents, or kits in identifying or screening female and male Momordica grosvenori plants.

[0065] The present application further provides the use of any of the above-described SNP markers, primers, detection reagents, or kits in marker-assisted breeding of Momordica grosvenori.

[0066] The present application further provides the use of any of the above-described SNP markers, primers, detection reagents, or kits in Momordica grosvenori hybrid breeding.

[0067] The detection method of the SNP marker of the present application is not specifically limited. Sequencing, single-strand conformation polymorphism polymerase chain reaction (PCR-SSCP), restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) and time-of-flight mass spectrometry and other technologies can all achieve SNP detection. Among them, sequencing is a detection technology with high accuracy, strong flexibility, high throughput and short detection cycle. It is only necessary to design a pair of primers on both sides of the SNP site, amplify a 400-700bp product, and then directly detect the genotype of the SNP site by sequencing.

[0068] The present application further provides a method for identifying female and male Momordica grosvenori plants, wherein the SNP marker for identifying female and male Momordica grosvenori plants is detected to predict whether the Momordica grosvenori plant is a female plant or a male plant, wherein the SNP marker is the SNP marker described in any one of the above items.

[0069] In a specific embodiment, the identification method comprises the following steps:

[0070] Extracting genomic DNA from the tested Momordica grosvenori;

[0071] Using the genomic DNA of the tested Momordica grosvenori as a template, PCR amplification is performed using primers to obtain PCR amplification products;

[0072] Detect the PCR amplification product. If the alleles at the X position of the diploid sequence of the amplified product are G and C, the Momordica grosvenori is a male plant; when the alleles at the X position of the diploid sequence of the Momordica grosvenori are C and C, the Momordica grosvenori is a female plant.

[0073] Example

[0074] Example 1 Acquisition of SNP markers

[0075] 1. Sample Collection and Processing

[0076] Over the past 20 years, Giffords has preserved 125 different varieties, including commercial and wild varieties collected in Guilin. This application sequenced these Momordica grosvenori germplasm resources, 102 of which were female and 17 were male. These plants were cultivated and the newly emerged shoots were selected for DNA extraction.

[0077] 2. DNA extraction and purification methods used the CTAB method, which is the most commonly used method for plant species.

[0078] 3. Screening and identification of SNP molecular markers:

[0079] a. High-throughput sequencing

[0080] The 125 Luo Han Guo varieties mentioned above were deeply sequenced using a high-throughput second-generation resequencing method. The average sequencing depth of each site reached 60×, and six million high MAF polymorphic sites were obtained.

[0081] At the same time, a male line of Luo Han Guo variety MFC0009 was subjected to third-generation sequencing including HIFI and Hi-C sequencing, and chromosome-level splicing was performed.

[0082] b. Genome assembly

[0083] After three generations of sequencing and assembly, the third-generation sequencing has completed the genome of the monk fruit and successfully assembled the genome at the chromosome level. The total length of the entire genome is 321Mbps.

[0084] In this field, it is currently not possible to achieve whole genome sequencing and chromosome splicing for Momordica grosvenori. This application is based on more than 100 Momordica grosvenori germplasm resources preserved by the applicant in the past 20 years. After a large amount of sequencing and splicing, the Momordica grosvenori genome was fully spliced ​​and the genome was successfully assembled to the chromosome level. This is the first time in this field. The total length of the entire genome is 321Mbps, and the spliced ​​genome and chromosomes are also determined for the first time by the applicant.

[0085] Although the cultivation history of Luo Han Guo has been hundreds of years, the complete sequence of its genome has not been fully assembled in the past 20 years. In 2018, Mian Xia et al. published an improved Luo Han Guo genome, Monk fruit (Qingpiguo) v1 Genome | CuGenDBv2 (cucurbitgenomics.org). Although 30,565 conserved protein regions can be found according to this genome, this genome is still composed of more than 4,000 contigs. This splicing has not reached the chromosome level. Therefore, it is difficult to locate any trait gene on this genome. The applicant used third-generation long sequencing technology and second-generation sequencing technology to sequence and splice a male commercial variety, and finally spliced ​​the Luo Han Guo genome.

[0086] c. GWAS association analysis of male and female traits

[0087] High-throughput next-generation sequencing was used to sequence the 125 Momordica grosvenori germplasm resources mentioned above, including 102 female plants and 17 male plants. SNP polymorphisms were analyzed across all accessions. A 6.4M locus SNP database was compiled. Using these SNPs and known male-female traits, genome-wide association analysis was performed on this database.

[0088] Genome-wide association analysis (GWAS) refers to the use of high-throughput sequencing technology to identify existing sequence variations within the entire genome, namely SNP typing, and the use of bioinformatics methods to screen out SNP sites associated with complex traits or measurable shapes. It is currently the most commonly used method for discovering functional gene markers.

[0089] The GLM+3pc method was used to calculate SNP associations in the GWAS analysis. The mixed linear model algorithm (Yu et al., 2006) was implemented in the rMVP R package v.1.0.6 (Yin et al., 2021). The kinship matrix for all Giffords breeds (calculated according to the method described in VanRaden, 2008) and the first three principal component variances (calculated as described above) were used as covariates in this analysis. Local linkage disequilibrium was calculated using Plink v.1.9 (Purcell et al., 2007).

[0090] GLM+3pc is a statistical model commonly used in genome-wide association studies (GWAS). It takes into account the effect of group structure on genetic variation and uses three principal components to correct population stratification and correct false positives. It is mainly used to correct the effect of group structure on genetic variation, thereby correcting false positives. In GWAS, a false positive refers to a situation where there is no real association but it is mistakenly identified as an association due to the influence of group structure. This situation may cause researchers to waste time and resources when looking for a real association. Compared to other methods for correcting population structure in GWAS, GLM+3pc is considered to be a relatively simple and computationally efficient method. It is also relatively easy to implement and explain, which is why this application chose this method for calculation.

[0091] The GWAS analysis revealed that the GWAS p-value (-log10(p)) for all SNPs within the continuous region of chromosome 3, starting at 0 and ending at 1 mbp, indicated that the male-female relationship was located at approximately 0.49 mbp. The Manhattan plot obtained from the GWAS analysis is shown in Figure 1, with the horizontal axis representing the base sequence from chromosomes 1 to 14 and the vertical axis representing -log10(p). 10 (p) represents the statistical significance value of GWAS analysis, that is, the p-value.

[0092] In GWAS analysis, the p-value is a measure of the statistical significance of the association between a gene or genetic variant and a trait. A smaller p-value indicates that the observed association is unlikely to be due to random factors, increasing the confidence in rejecting the null hypothesis. However, the p-value only provides information about the association. Although SNPs with a (-log10(p)) greater than or equal to 8 can theoretically be considered to be significantly associated with male and female traits, this application detected SNPs with a (-log10(p)) greater than or equal to 25 as the sites most closely associated with male and female traits. Selecting any of these sites can distinguish between male and female plants.

[0093] The threshold is selected between (-log 10At loci with a p-value greater than or equal to 25, the SNPs associated with sex traits on chromosome 3 are located within the peaks outlined in the figure above. These SNPs form a set of male and female haploid SNPs. Any of these SNPs can be used to distinguish male and female plants, and these SNPs are called golden SNPs.

[0094] At the same time, this application also conducted a statistical analysis on the GWAS analysis results. On chromosome 3, there are 471 SNP sites with p-vlaue values ​​greater than 25 and associated with sex traits. Any of these 471 SNP sites can be used to distinguish between male and female plants. In addition, this application found that the larger the p-vlaue value, the higher the accuracy of its use in distinguishing between male and female plants. By conducting a statistical analysis on the GWAS analysis results, there are 9 SNP sites on chromosome 3 with p-vlaue values ​​greater than 30 and associated with sex traits. These 9 SNP sites have a higher accuracy in distinguishing between male and female plants.

[0095] By comparing the actual SNP p-value to the quantile of the probability distribution of the probability value / expected value, we can compare two p-value probability distributions. As shown in Figure 2, the Y-axis shows the actual SNP p-value, or observed, and the X-axis shows the probability value / expected value of the uniform distribution, or expected. All of these indicators are converted to -log10.

[0096] Figure 2 shows the distribution of P values ​​associated with male-female traits obtained from statistical tests in genome-wide association studies (GWAS) of male-female relationships. The expected P value is calculated under the null hypothesis and plotted on the x-axis, while the observed P value is plotted on the y-axis. A straight line with a slope of one indicates that the observed P value distribution conforms to the null hypothesis, while a curved line indicates deviation from the null hypothesis. The greater the deviation, the higher the probability of association. For example, in Figure 2, loci with a -log10(p) greater than 30 have the highest probability of being associated with male-female traits.

[0097] d. Screening of SNP molecular markers in Momordica grosvenori

[0098] From the SNP sites with p-vlaue values ​​greater than 30 and associated with sex traits, a site with high p-vlaue was further screened, which also met the requirements of allele-specific PCR designed as a fluorescent endpoint method. The sequence of the SNP marker site is shown in SEQ ID NO: 1:

[0099] The nucleotide represented by S at position 122 in SEQ ID NO: 1 is G or C, which is marked as [G / C], i.e., a SNP marker site. The polymorphism of the sequence in female and male plants is that when the alleles at this position in the diploid Momordica grosvenori are G and C, the Momordica grosvenori is a male plant, and when the alleles at this position in the diploid Momordica grosvenori are C and C, the Momordica grosvenori is a female plant, i.e.

[0100] G:C is male,

[0101] C: C is female.

[0102] Example 2 Verification and application of SNP molecular markers

[0103] The above marks were verified in 2019, 2020 and 2021 respectively. The verification methods used are as follows

[0104] 1. Cultivate seedlings and select tender shoots for DNA extraction.

[0105] 2. DNA extraction and purification methods used the CTAB method, which is the most commonly used method for plant species.

[0106] 3. Amplify the nucleotide fragment containing the SNP site and detect the amplification of specific primers.

[0107] The primers for allelic PCR of SEQ ID NO: 1 using the fluorescent endpoint method were designed as follows:

[0108] Forward primer 1 (SEQ ID NO: 2):

[0109] 5'-GAAGGTGACCAAGTTCATGCTTCAACCATTGGGGATGGAG-3';

[0110] Forward primer 2 (SEQ ID NO: 3):

[0111] 5'-GAAGGTCGGAGTCAACGGATTTCAACCATTGGGGATGGAC-3';

[0112] Reverse primer (SEQ ID NO: 4):

[0113] 5'-GTAGAAGCGGCTGCTTTTACTGG-3'.

[0114] The three primers were mixed in the following proportions: 12 μl of forward primer 1, 12 μl of forward primer 2, 30 μl of reverse primer, and 46 μl of water to form 100 μl of primer mixture.

[0115] The primer mixture (0.07 μl), DNA template (2.5 μl), and fluorescent PCR mixture (2.5 μl) were mixed and placed in a fluorescent PCR instrument. The reaction was carried out according to the following cycling parameters: (1) Denaturation: 94°C, 15 min, 1 cycle; (2) Specific cycle: 94°C, 20 seconds, 61°C-55°C, decreasing by 0.6°C, 10 cycles; (3) Amplification cycle: 94°C, 20 seconds, 55°C, 60 seconds, 26 cycles; (4) Read cycle: 30°C, 60 seconds, 1 cycle. The nucleotide fragment where the SNP to be detected is amplified.

[0116] 4. High-throughput sequencing

[0117] After the PCR reaction was completed, the nucleotide fragments of the amplified SNPs of the 125 Siraitia grosvenori varieties were subjected to high-throughput DNA testing using the second-generation sequencing method to achieve early detection of male and female plants. The results are shown in Table 1 below.

[0118] 2020: The same verification method as in 2019 was used, with a total of 194 samples. The results are shown in Table 1 below.

[0119] 2021: The same verification method as in 2019 was used, with a total sample size of 383. The results are shown in Table 1 below.

[0120] Table 1

[0121] The experimental seedlings from 2019 to 2021 were tested, and as can be seen from the verification results in Table 1, the data confirmed that the prediction accuracy of this marker is more than 94.8%.

[0122] Momordica grosvenori is a perennial vine, and the breeding of seedlings requires 2-3 years. This application has expended a great deal of time and manpower on the process of collection, cultivation, testing, and revalidation. The development of this marker has expanded the scale of breeding from the selection of dozens of plantations to thousands, increasing the scale of breeding selection by several dozen times and greatly improving the efficiency of breeding. The applicants performed these sequencing, planting, and final validation processes as accurately as possible, ultimately achieving a high prediction accuracy. This demonstrates that the method of this application has both high accuracy and high efficiency.

[0123] This application verified the SNP marker of this application in 2019, 2020 and 2021 respectively. For three consecutive years, this marker provided stable, fast and accurate predictions. Such a long verification process is very rare in this field because it requires a lot of experimental means and manpower and material resources. The accuracy of the SNP marker provided by this application has reached 96.1% during the verification process in 2019. This is the first time in this field that the accuracy of male and female traits has been rapidly detected. However, this application has been rigorous and serious and has conducted two more years of verification, at the expense of a lot of manpower and material resources. Facts have proved that the accuracy of the SNP marker provided by this application is very high, which is a result that has not been achieved in this field so far.

[0124] Although the embodiments of the present application are described above, the present application is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are merely illustrative and instructive, and not restrictive. A person of ordinary skill in the art, under the guidance of this specification and without departing from the scope of protection of the claims of this application, can also make many forms, all of which fall within the scope of protection of this application.

Claims

1. SNP markers for identifying female and male Siraitia grosvenorii plants, wherein, The SNP marker is a locus with a p-value greater than 25.

2. The SNP marker according to claim 1, wherein the nucleotide sequence of the SNP marker is as shown in SEQ ID NO:1, and the position is the 122nd position of the nucleotide sequence, wherein the nucleotide represented by S in SEQ ID NO:1 is G or C.

3. The SNP marker according to claim 1 or 2, wherein When the alleles at the S position of the diploid Momordica grosvenori are G and C, the Momordica grosvenori is a male plant; when the alleles at the S position of the diploid Momordica grosvenori are C and C, the Momordica grosvenori is a female plant.

4. Primers for detecting the SNP marker according to any one of claims 1 to 3, including Forward primer 1: 5’-GAAGGTGACCAAGTTCATGCTTCAACCATTGGGGATGGAG-3’; Forward primer 2: 5’-GAAGGTCGGAGTCAACGGATTTCAACCATTGGGGATGGAC-3’; Reverse primer: 5’-GTAGAAGCGGCTGCTTTTACTGG-3’.

5. A detection reagent or kit for detecting the SNP marker according to any one of claims 1 to 3, wherein, Comprising the primers according to claim 4.

6. Use of the SNP marker according to any one of claims 1 to 3, the primer according to claim 4, or the detection reagent or kit according to claim 5 in identifying or screening female and male Momordica grosvenori plants.

7. Use of the SNP marker according to any one of claims 1 to 3, the primer according to claim 4, or the detection reagent or kit according to claim 5 in marker-assisted breeding of Momordica grosvenori.

8. Use of the SNP marker according to any one of claims 1 to 3, the primer according to claim 4, or the detection reagent or kit according to claim 5 in cross-breeding of Momordica grosvenori.

9. A method for identifying female and male Siraitia grosvenorii plants, wherein, By detecting the SNP marker for identifying female and male Momordica grosvenori plants of the Momordica grosvenori to be tested, predicting whether the Momordica grosvenori is a female or male plant, wherein the SNP marker is the SNP marker according to any one of claims 1 to 3.

10. The method according to claim 9, wherein, Including Extracting the genomic DNA of the Momordica grosvenori to be tested; Using the genomic DNA of the Momordica grosvenori to be tested as a template and performing PCR amplification with the primers according to claim 4 to obtain a PCR amplification product; Detecting the PCR amplification product. If the alleles at the S position of the amplified product sequence in the diploid are G and C, the Momordica grosvenori is a male plant; when the alleles at the S position of the diploid Momordica grosvenori are C and C, the Momordica grosvenori is a female plant.

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