A rdp molecular marker primer for identifying the gender of momordica grosvenori, a kit and application thereof

Abstract

The application discloses a RAPD molecular marker primer for identifying the gender of Momordica grosvenori. The nucleotide sequence of the RAPD molecular marker primer is ACAGCCTGCT. The application discloses a kit containing the RAPD molecular marker primer for identifying the gender of Momordica grosvenori and application thereof. The application also discloses a method for identifying the gender of Momordica grosvenori by using the RAPD molecular marker primer. The method comprises the following steps: performing PCR amplification on total DNA of Momordica grosvenori by using the RAPD molecular marker primer, performing electrophoresis to obtain corresponding bands, and then identifying the gender of Momordica grosvenori. The RAPD molecular marker primer provided by the application is a random primer, has high specificity for male plants, can efficiently and rapidly distinguish male plants from female plants of Momordica grosvenori, and has the advantages of small DNA dosage, low purity requirement, simple experimental process, fast working progress and low cost in the identification test.

Description

Technical Field

[0001] This invention relates to RAPD molecular marker technology, and more specifically, to a RAPD molecular marker primer, kit, and application for identifying the sex of monk fruit. Background Technology

[0002] Monk fruit (Siraitia grosvenorii) is a species of cucurbitaceae plant endemic to my country, possessing both edible and medicinal properties. It has effects such as clearing heat and moistening the lungs, relieving cough and phlegm, and promoting bowel movements. Monk fruit is a dioecious plant, and currently cultivated varieties require artificial pollination to produce fruit. Its seeds are heterozygous, with male plants comprising 70-80% and female plants 20-30% after germination. With market demand, there is an urgent need for superior monk fruit varieties. However, there is still a certain time difference between male and female monk fruit plants from growth to flowering, and there is no obvious difference before flowering. Therefore, finding molecular markers related to the sex of monk fruit is of great significance both in production and in genetic breeding.

[0003] Patent application ZL201310108992.5 discloses a SCAR molecular marker for identifying the sex of *Monk Fruit* (Siraitia grosvenorii), which distinguishes between male and female *Monk Fruit* through DNA extraction, primer design, PCR reaction, and detection steps. The method first uses random primer P7 to amplify a male-specific band. However, in experiments using the FN (female) and FNM (male) gene pools as templates, no target band appeared when amplified with P7. Secondly, the patent uses the *Monk Fruit* genome as a template and primers Z1200-F1 and Z1200-R1 for amplification. Male plants showed a specific fragment amplified, with a target fragment size of 648 bp. However, in experiments using single male and female plants as templates, when amplified with Z1200-F1 and Z1200-R1, male plants showed a specific band at approximately 250 bp, and female plants showed a specific band at approximately 200 bp. However, the overall amplification effect was poor and cannot be used as an effective tool for identifying the sex of *Monk Fruit* plants.

[0004] Patent No. 201811180050.7 discloses a method for rapid identification of male and female *Siraitia grosvenorii* plants based on specific molecular markers. This method first uses random primers RP2 and RP8 to amplify a male-specific fragment, yielding a sequence approximately 1400 bp in length. However, in experiments using the FN and FNM gene pools as templates, RP2 did not amplify a sex-specific band, and RP8 did not produce any target band. Secondly, using the *Siraitia grosvenorii* genome as a template, primers DM3F and DM3R amplified a specific fragment from male plants, with a target fragment size of 463 bp. However, in experiments using FN, FNM, LU, and LUM individual plants as templates, amplification with DM3F and DM3R did not yield satisfactory results.

[0005] In summary, neither of the SCAR primers and identification methods mentioned in the two patents can amplify male-specific bands and thus cannot identify the sex of Luo Han Guo. Therefore, it is necessary to provide a molecular marker primer and identification method that can amplify male-specific bands to identify the sex of Luo Han Guo. Summary of the Invention

[0006] One objective of this invention is to provide a RAPD molecular marker primer for identifying the sex of monk fruit. The selected RAPD molecular marker primer has high specificity for male monk fruit plants and can amplify specific bands that are significantly different from those of female plants, thereby enabling the identification of the sex of monk fruit.

[0007] One of the technical solutions to achieve the purpose of this invention is: RAPD molecular marker primers for identifying the sex of monk fruit, with the nucleotide sequence: ACAGCCTGCT.

[0008] A second objective of this invention is to provide a kit for identifying the sex of monk fruit.

[0009] Specifically, a kit containing RAPD molecular marker primers for identifying the sex of monk fruit is provided, wherein the nucleotide sequence of the RAPD molecular marker primers is: ACAGCCTGCT.

[0010] A third objective of this invention is to provide the application of the aforementioned RAPD molecular marker primers for identifying the sex of monk fruit.

[0011] Specifically, this paper provides the application of the aforementioned RAPD molecular marker primers for identifying the sex of monk fruit in the preparation of SCAR primers.

[0012] The fourth objective of this invention is to provide a method for identifying the sex of monk fruit using the RAPD molecular marker primers described above.

[0013] Specifically, the method for identifying the sex of monk fruit using the aforementioned RAPD molecular marker primers includes the following steps:

[0014] (1) Extract total DNA from the organs and tissues of monk fruit;

[0015] (2) Take the RAPD molecular marker primers and the total DNA for PCR amplification reaction;

[0016] (3) Perform electrophoresis on the PCR product obtained in step (2). If a specific strong band appears near 2400bp in the obtained electrophoretic band, it is a male Luo Han Guo plant.

[0017] In step (1), the organs of the monk fruit include leaves, fruit, flowers, roots or stems.

[0018] In step (2), the PCR system consists of 25 μL of ddH2O, 17 μL of total DNA from monk fruit, 1 μL of dNTPs, 2 μL of RAPD molecular marker primers, and 3 μL of Ex Taq polymerase.

[0019] In step (2), the PCR reaction conditions are: 95℃ for 5 min, then 95℃ for 30 sec, 35℃ for 30 sec, 72℃ for 30 sec, repeated 30 times, and finally 72℃ for 10 min.

[0020] Beneficial effects

[0021] 1. The RAPD molecular marker primers for sex determination of monk fruit provided by this invention are highly specific for male monk fruit plants and can amplify specific bands that are significantly different from those of female plants, thus efficiently distinguishing the sex of monk fruit.

[0022] 2. The RAPD molecular marker primers provided by this invention are random primers, without strict species boundaries, and have broad applicability and universality. They do not require prior knowledge of genomic information and can quickly and easily identify the sex of monk fruit.

[0023] 3. The RAPD experiment of this invention uses very little DNA, has low purity requirements, is simple in process, fast in progress and low in cost. Attached Figure Description

[0024] Figure 1 This image shows the nucleic acid electrophoresis images of eight primers (including specific and non-specific primers) used in the screening of specific primers.

[0025] Wherein: F represents female plants, M represents male plants, and MW represents molecular weight markers. The selected male and female samples of Luo Han Guo were all from the same maternal parent.

[0026] Figure 2The images show the nucleic acid electrophoresis results of DNA amplification using primer RP3 from eight individual male and female *Monk Fruit* plants. The selected male and female *Monk Fruit* plants were all derived from different maternal parents. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] I. Screening of Specific RAPD Primers

[0029] Using the total DNA from male and female *Siraitia grosvenorii* plants as a template, more than 1,000 primers from the RAPD random primer library were used for amplification. Polymorphic bands with good repeatability and clear bands were selected, and the specificity of the primers was verified by individual male and female plants.

[0030] (1) Total DNA extraction from Luo Han Guo seedlings

[0031] Leaves from both female and male *Monk Fruit* plants were collected, and 0.1g of each leaf was placed in a 2mL EP tube containing glass beads. After pre-freezing in liquid nitrogen, the EP tubes were placed in a grinder to grind the plant samples into powder. The plant genomic DNA rapid extraction kit (purchased from Baori Biotechnology (Beijing) Co., Ltd., kit number: 9768) was used to extract total DNA from female and male *Siraitia grosvenorii* plants. The obtained total DNA from the leaves of female and male *Siraitia grosvenorii* plants was stored at -20℃.

[0032] (2) Establishment of male and female cluster gene pools

[0033] Six portions of female plant leaf genomic DNA were mixed in equal amounts to form a female plant cluster gene pool, and six portions of male plant leaf genomic DNA were mixed in equal amounts to form a male plant cluster gene pool.

[0034] (3) PCR reaction

[0035] More than 1000 primers from the RAPD primer library were used to perform PCR amplification and screening of sex-related RAPD molecular marker primers in both female and male gene pools. The specific reaction system is as follows:

[0036] The PCR system consisted of 25 μL of the following components: 17 μL of ddH2O, 2 μL of total DNA from monk fruit, 1 μL of dNTPs, 2 μL of RAPD primers, and 3 μL of ExTaq polymerase.

[0037] The reaction conditions were: 95℃ for 5 min, then 95℃ for 30 sec, 35℃ for 30 sec, 72℃ for 30 sec, repeated 30 times, and finally 72℃ for 10 min.

[0038] (4) Electrophoresis of PCR products

[0039] The PCR amplification products obtained in step (3) were electrophoresed in a 1% agarose gel with a sample volume of 10 μL per lane, a voltage of 80 V, and a time of 60 min.

[0040] (5) Comparison of band differences

[0041] Among more than 1000 primers, primer RP3 was selected, which amplified bands in male and female samples that showed significant differences. Figure 1 During the screening process, nucleic acid electrophoresis images were obtained by amplifying the samples using eight RAPD random primers. The images clearly show that there is no difference in the nucleic acid electrophoresis bands of male and female plants of RP1, RP2, RP4, RP5, RP6, RP7 and RP8. Only the female sample of Luo Han Guo amplified by RP3 lacks a bright band around 2400bp compared to the male sample.

[0042] The eight RAPD primer sequences are as follows:

[0043] RP1: GTGGCATCTC;

[0044] RP2: CATCGCCGCA;

[0045] RP3: ACAGCCTGCT;

[0046] RP4: GGCTGCAATG;

[0047] RP5: GGCTGCGACA;

[0048] RP6: AAGGCTCACC;

[0049] RP7: AGAGCCGTCA;

[0050] RP8: AGGCAGAGCA.

[0051] II. Primer Specificity Verification

[0052] To further determine whether the RAPD polymorphic bands screened from male and female DNA clusters are sex-linked, DNA from eight different male and female plants was extracted, and RP3 was validated to confirm that it exhibits stable specificity in different individual Luo Han Guo plants.

[0053] (1) Total DNA extraction from Luo Han Guo seedlings

[0054] Eight groups of male and female plants from different sources were selected. 0.1g of leaves from each plant were weighed and placed in 2mL EP tubes containing glass beads. After pre-freezing in liquid nitrogen, the EP tubes were placed in a grinder to grind the plant samples into powder. The plant genomic DNA rapid extraction kit was used to extract genomic DNA from each individual Luo Han Guo plant. The total DNA obtained from the Luo Han Guo leaves was stored at -20℃.

[0055] (2) PCR reaction

[0056] PCR amplification was performed using the following primers: RP3: ACAGCCTGCT.

[0057] The PCR system consisted of 25 μL of the following components: 17 μL of ddH2O, 2 μL of total DNA from monk fruit, 1 μL of dNTPs, 2 μL of RAPD primers, and 3 μL of ExTaq polymerase.

[0058] The reaction conditions were: 95℃ for 5 min, then 95℃ for 30 sec, 35℃ for 30 sec, 72℃ for 30 sec, repeated 30 times, and finally 72℃ for 10 min.

[0059] (3) Electrophoresis of PCR products

[0060] The PCR amplification products obtained in step (2) were electrophoresed in a 1% agarose gel with a sample volume of 10 μL per lane, a voltage of 80 V, and a time of 60 min.

[0061] (4) Comparison of band differences

[0062] Comparative electrophoresis imaging analysis revealed that when using primer RP3 for PCR amplification, female *Siraitia grosvenorii* plants (lanes 1-8) lacked a specific strong band at approximately 2400 bp compared to male plants (lanes 9-16). Specifically... Figure 2 As shown, there are significant differences between the two groups. Female *Siraitia grosvenorii* plants (lanes 1-8) lack a specific strong band around 2400 bp compared to male plants (lanes 9-16). The polymorphism amplified by RP3 is stable across female and male plant clusters, as well as among individual male and female plants, and the specific band is clear. This indicates that it is a sex-linked RAPD molecular marker with good reproducibility, and can be used as a RAPD molecular marker for sex identification in *Siraitia grosvenorii*.

[0063] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention, and these will not affect the effectiveness and practicality of the present invention. sequence list <110> Guilin Rhein Biotechnology Co., Ltd. <120> A RAPD molecular marker primer, kit, and application for identifying the sex of monk fruit. <140> 2022100434202 <141> 2022-01-14 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <400> 1 gtggcatctc 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <400> 2 catcgccgca 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <400> 3 acagcctgct 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <400> 4 ggctgcaatg 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <400> 5 ggctgcgaca 10 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <400> 6 aaggctcacc 10 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <400> 7 agagccgtca 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <400> 8 aggcagagca 10

Claims

1. A method for identifying the sex of monk fruit using RAPD molecular marker primers, characterized in that, Includes the following steps: (1) Extract total DNA from organs and tissues of monk fruit; (2) Take the RAPD molecular marker primer and perform PCR amplification reaction with the total DNA. The nucleotide sequence of the RAPD molecular marker primer is: ACAGCCTGCT; (3) Perform electrophoresis on the PCR product obtained in step (2). If a specific strong band appears near 2400bp in the obtained electrophoretic band, it is a male Luo Han Guo plant.

2. The method for identifying the sex of *Siraitia grosvenorii* using RAPD molecular marker primers according to claim 1, characterized in that, In step (1), the organs of the monk fruit include leaves, fruit, flowers or stems.

3. The method for identifying the sex of *Siraitia grosvenorii* using RAPD molecular marker primers according to claim 1, characterized in that, In step (2), the PCR system consists of 25 μL of ddH2O, 17 μL of total DNA from monk fruit, 1 μL of dNTPs, 2 μL of RAPD molecular marker primers, and 3 μL of Ex Taq polymerase.

4. The method for identifying the sex of *Siraitia grosvenorii* using RAPD molecular marker primers according to claim 3, characterized in that, In step (2), the PCR reaction conditions are: 95℃ for 5 min, then 95℃ for 30 sec, 35℃ for 30 sec, 72℃ for 30 sec, repeated 30 times, and finally 72℃ for 10 min.

Images