Molecular marker and reagent for identifying a full hermaphrodite line trait of a melon

Abstract

The application discloses a molecular marker and reagent for identifying a melon full hermaphrodite line character. The application finds that a melon flower sex molecular marker is related to the sex of melon flowers, and the melon flower sex molecular marker is the 30th nucleotide corresponding to SEQ ID No. 1 in a sequence list in a melon genome, which is C or T. Experiments prove that the melon flower sex molecular marker of the application can be used for identifying the character of melon flowers, and the melon flower sex molecular marker of the application can be used for molecular marker assisted breeding, and the genotype of full hermaphrodite flowers can be selected for the production of melons. The application has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and relates to a molecular marker and reagent for identifying a melon hermaphrodite line trait. BACKGROUND

[0002] Melon is widely cultivated all over the world, is the earliest melon as fruit in China, and is an important horticultural crop, which has important economic value.

[0003] At present, melon varieties cultivated commercially are mostly androecious hermaphrodite plants, and the main branches open androecious flowers and the lateral branches open hermaphrodite flowers at low nodes. The fruits of androecious hermaphrodite melon plants are developed from hermaphrodite flowers, and the number of hermaphrodite flowers on lateral branches is limited. After flowering, melon is often affected by pollination, nutritional conditions and other conditions, and the yield of melon is limited. Therefore, improving the new planting mode of using hermaphrodite line as the main cultivation plant and mixed planting with androecious plants and using bumblebee pollination is of great significance to improve the fruit setting rate of melon, increase the yield and reduce the cost of melon planting. It is of great significance to obtain and identify melon hermaphrodite line.

[0004] Using the molecular marker closely linked to the target gene, the molecular marker assisted selection breeding can improve the selection efficiency and accuracy of the target trait, accelerate the breeding process and expand the application range of the target gene, which is a necessary condition for the molecular marker assisted selection breeding. Therefore, developing the molecular marker closely linked to the hermaphrodite trait has important practical value for improving the efficiency of breeding hermaphrodite parent, expanding the application range of hermaphrodite gene and promoting the stable development of melon industry. SUMMARY

[0005] The technical problem to be solved by the present application is how to identify the melon flower sex trait.

[0006] To solve the above technical problem, the present application first provides the application of a melon flower sex molecular marker or a substance for detecting the melon flower sex molecular marker in identifying or assisting in identifying the melon flower sex trait;

[0007] The melon flower sex molecular marker is the 30th nucleotide corresponding to SEQ ID No. 1 in the sequence listing, and the melon flower sex molecular marker is C or T.

[0008] In the above application, the substance for detecting the melon flower sex molecular marker can include a primer pair capable of amplifying the melon flower sex molecular marker, and the primer pair is composed of single-stranded DNAs with names P1 and P2, respectively. The P1 is a single-stranded DNA specifically combined with the upstream of the 30th nucleotide of SEQ ID No. 1 in the melon genome, and the P2 is a single-stranded DNA reversely specifically combined with the downstream of the 30th nucleotide of SEQ ID No. 1 in the melon genome.

[0009] In the application, the sequence of P1 can be SEQ ID No. 3 in the sequence listing; and the sequence of P2 can be SEQ ID No. 4 in the sequence listing.

[0010] In the application, the substance for detecting the melon flower sex molecular marker can further include a restriction enzyme SacI.

[0011] In the application, the substance for detecting the melon flower sex molecular marker can be the primer pair, and can further consist of the primer pair and the restriction enzyme SacI.

[0012] The application further provides a method for detecting a melon flower sex trait, which comprises: using genomic DNA of a melon to be detected as a template, performing PCR amplification by using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing, performing enzyme digestion of the obtained PCR product by using a restriction enzyme SacI, and detecting the size of the enzyme digestion product; a homozygous melon with enzyme digestion products of two DNA fragments of 29 and 166 bp is or is a candidate for a melon with male flowers on the main branch and bisexual flowers on the low-order nodes of the lateral branches; a homozygous melon with an enzyme digestion product of one DNA fragment of 195 bp is or is a candidate for a melon with bisexual flowers on the main branch and the lateral branches; and a heterozygous melon with enzyme digestion products of three DNA fragments of 29, 166 and 195 bp is or is a candidate for a melon with male flowers on the main branch and bisexual flowers on the low-order nodes of the lateral branches.

[0013] In the application, the melon with male flowers on the main branch and bisexual flowers on the low-order nodes of the lateral branches refers to a melon with bisexual flowers on the first to third nodes of the lateral branches at the base and male flowers on the fourth node and above.

[0014] The application further provides a melon breeding method, which comprises: using genomic DNA of a melon to be detected as a template, performing PCR amplification by using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing, performing enzyme digestion of the obtained PCR product by using a restriction enzyme SacI, and selecting the melon to be detected with an enzyme digestion product of one DNA fragment of 195 bp as a parent for breeding.

[0015] The application further provides a method for producing a melon, which comprises: using genomic DNA of a melon to be detected as a template, performing PCR amplification by using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing, performing enzyme digestion of the obtained PCR product by using a restriction enzyme SacI, and selecting the melon to be detected with an enzyme digestion product of one DNA fragment of 195 bp for producing a melon.

[0016] In the above, the reaction system for PCR amplification using the primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing can be: 2xGoTaq Mix (promega, M712B) 7 μL, the P1 1 μL, the P2 1 μL, the genomic DNA of the melon to be tested, and 20 μL of sterile ultrapure water. The concentration of the P1 and the P2 in the system is 0.5 μM.

[0017] The reaction conditions for PCR amplification using the primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing can be: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 15 s, 35 cycles, and 72℃ extension for 5 min.

[0018] The substance for detecting the melon flower sex molecular marker also belongs to the protection scope of the present application.

[0019] The application of the substance for detecting the melon flower sex molecular marker in preparing or assisting in preparing the product for identifying the melon flower sex trait also belongs to the protection scope of the present application.

[0020] The application of the substance for detecting the melon flower sex molecular marker in melon breeding also belongs to the protection scope of the present application.

[0021] The application of the melon flower sex molecular marker in identifying or assisting in identifying the melon flower sex trait also belongs to the protection scope of the present application.

[0022] The application of the melon flower sex molecular marker in melon breeding also belongs to the protection scope of the present application.

[0023] In the present application, the melon flower traits are all the traits of flowers under natural growth.

[0024] Experiments prove that the melon flower sex molecular marker of the present application can be used for identifying the traits of melon flowers, and the melon flower sex molecular marker of the present application can be used for molecular marker assisted breeding, and the genotype of the whole bisexual flower can be selected for the production of melon. The present application has a good application prospect.

[0025] The present application is further described in detail below in conjunction with specific embodiments. The examples provided below are only for illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0026] Biological material preservation instructions

[0027] Abbreviation of depositary institution: CGMCC

[0028] Name of depositary institution: General Microbiological Center of China

[0029] Address of depositary institution: No. 1, Yihuan 3rd Road, Beijing City, Chaoyang District, China, Postcode: 100101

[0030] Date of deposit: May 08, 2023

[0031] Registration number of deposit center: CGMCC No. 26394

[0032] Classification name: Cucumis melo

[0033] Plant number: 13C-WT

[0034] Biological material preservation instructions

[0035] Abbreviation of depositary institution: CGMCC

[0036] Name of depositary institution: General Microbiological Center of China

[0037] Address of depositary institution: No. 1, Yihuan 3rd Road, Beijing City, Chaoyang District, China, Postcode: 100101

[0038] Date of deposit: May 08, 2023

[0039] Registration number of deposit center: CGMCC No. 26395

[0040] Classification name: Cucumis melo

[0041] Plant number: 13C-68 BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The electrophoresis result of the enzyme digestion product. The first lane on the left is a DNA molecular weight marker. DETAILED DESCRIPTION

[0043] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified. In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged. In the following examples, unless otherwise specified, the 1st nucleotide of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last nucleotide is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0044] The melon 13C inbred line (13C-WT) in the following examples was donated by Wang Huaisheng (Beijing, China), which has main branches with male flowers and lateral branches with hermaphrodite flowers at low nodes (i.e., the first to third nodes from the base of the lateral branches have hermaphrodite flowers, and the fourth node and above have male flowers). The 13C-WT was deposited with the China General Microbiological Culture Collection Center on May 8, 2023, and the deposit number is CGMCC No. 26394.

[0045] The 13C-68 in the following examples was obtained by EMS mutagenesis of the melon 13C-WT by Chai Sen (Shandong Province, China) in December 2020. The 13C-68 is a full hermaphrodite flower line (i.e., all flowers are hermaphrodite flowers). The 13C-68 was deposited with the China General Microbiological Culture Collection Center on May 8, 2023, and the deposit number is CGMCC No. 26395.

[0046] The kit for detecting the melon flower sex trait in Example 1 can identify the full hermaphrodite flower trait of melon

[0047] The present example provides a molecular marker related to melon flower sex, which is the 30th nucleotide of SEQ ID No. 1 in the sequence listing, denoted as a melon flower sex molecular marker. The molecular marker is C or T in the melon genomic DNA, and the upstream and downstream sequences containing the molecular marker in the melon genomic DNA are SEQ ID No. 1 or SEQ ID No. 2.

[0048] The genotype of melon is defined according to whether the melon flower sex molecular marker in the genomic DNA is C or T. The AA genotype corresponds to both homologous chromosomes of SEQ ID No. 1, the Aa genotype corresponds to one of SEQ ID No. 1 and the other of SEQ ID No. 2, and the aa genotype corresponds to both homologous chromosomes of SEQ ID No. 2. Among them, the AA genotype and the aa genotype can be stably inherited.

[0049] A kit for detecting the melon flower sex trait is designed according to the melon flower sex molecular marker. The kit consists of a primer pair named A1 and a SacI endonuclease. A1 consists of single-stranded DNA named P1 and P2. P1 is a single-stranded DNA represented by SEQ ID No. 3, and P2 is a single-stranded DNA represented by SEQ ID No. 4. The sequences are as follows:

[0050] P1: 5'-ATTATAATCCTTCTTCTTCTTCTC G AG CT-3' (SEQ ID No. 3 in the sequence listing, underlined is the mutation nucleotide introduced by dCAPs marker);

[0051] P2: 5'-TATTAGAAGAAGAGGAAGGAGTTGGAAGCC-3' (SEQ ID No. 4 in the sequence listing).

[0052] 1. The melons to be identified are as follows:

[0053] 50 plants of each melon were selected for testing:

[0054] P1: melon 13C-WT;

[0055] P2: 13C-68;

[0056] F1: hybrid F1 of melon 13C-WT and 13C-68;

[0057] F2: hybrid F2 of melon 13C-WT and 13C-68 (F2, i.e. the selfed generation of F1).

[0058] 2. Identification of the full hermaphrodite flower trait of melon

[0059] The genomic DNA of the tender leaves of each melon above was extracted by CTAB method, and primer pair A1 was used for PCR amplification. The 20 μL PCR amplification reaction system was: 2xGoTaq Mix (promega, M712B) 10 μL, P1 1.0 μL, P2 1.0 μL, melon genomic DNA 50 ng, and sterile ultrapure water was added to 20 μL. The PCR amplification reaction conditions in the PCR instrument were: 95°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 15 s, 35 cycles, 72°C extension for 5 min.

[0060] After PCR amplification, the obtained PCR reaction product was digested with SacI, and the 14 μL digestion reaction system was: PCR reaction product 7 μL, Cutsmart buffer 1.4 μL, SacI 0.2 μL, ddH2O 5.4 μL, 37°C digestion for 6 hours. Then the digestion product was electrophoresed on a 4.0% agarose gel at 120 V for 50 min, and after M5 Gelred Plus nucleic acid staining, it was observed and recorded under the gel imaging system. Cutsmart buffer and SacI are products of NEB company.

[0061] The results show that the enzyme digestion products have three DNA fragments, the electrophoresis results show that the Aa genotype melon has three bands, the sizes of the bands are 195bp, 29bp and 166bp; the enzyme digestion products have one DNA fragment, the electrophoresis results show that the aa genotype melon has one band, the size of the band is 195bp; the enzyme digestion products have two DNA fragments, the electrophoresis results show that the AA genotype melon has two bands, the sizes of the bands are 29bp and 166bp. The sequences of the PCR reaction products and the enzyme digestion products are consistent with the expected sequences. The electrophoresis results of some plants are shown in Figure 1

[0062] The sex of the flowers of each plant is observed, and the results show that the AA and Aa genotype melons have male flowers on the main branches and hermaphroditic flowers on the low-order lateral branches, and the aa genotype melon has hermaphroditic flowers on the main branches and the lateral branches, and part of the results are shown in Table 1.

[0063] Table 1, the corresponding genotype and phenotype of the detected melon

[0064]

[0065]

[0066] In Table 1, the hermaphroditic flowers on the low-order lateral branches refer to the hermaphroditic flowers on the 1st-3rd nodes from the base of the lateral branches, and the male flowers on the 4th node and above.

[0067] It is shown that the melon flower sex molecular marker and the complete set of reagents for identifying or assisting in identifying the melon flower sex trait of the present application can identify the melon flower sex, and can be used for molecular marker assisted breeding.

[0068] SEQ ID No. 1 and SEQ ID No. 2 involved in the above examples are as follows:

[0069] SEQ ID No. 1:

[0070]

[0071] SEQ ID No. 2:

[0072]

[0073] ​The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of a melon flower sex molecular marker or a substance for detecting the melon flower sex molecular marker in identifying the sex traits of melon flowers; The melon flower sex molecular marker is the 30th nucleotide corresponding to SEQ ID No. 1 in the sequence list in the melon genome, and the melon flower sex molecular marker is C or T; The flower sex is as follows: the main branches have male flowers, the side branches have bisexual flowers at low nodes, and the main branches and side branches all have bisexual flowers; in, Male flowers bloom on the main branches and bisexual flowers bloom on the lower nodes of the side branches means that bisexual flowers bloom on the 1st to 3rd nodes from the base of the side branches, and male flowers bloom on the 4th node and above.

2. The use according to claim 1, characterized in that: The substance for detecting the melon flower sex molecular marker includes a primer pair that can amplify the melon flower sex molecular marker, and the primer pair is composed of single-stranded DNA named P1 and P2, respectively. The P1 is a single-stranded DNA that specifically binds to the 30th position upstream of SEQ ID No.1 in the melon genome, and the P2 is a single-stranded DNA that specifically binds to the 30th position downstream of SEQ ID No.1 in the melon genome in the opposite direction.

3. The use according to claim 2, characterized in that: The sequence of P1 is SEQ ID No. 3 in the sequence listing; the sequence of P2 is SEQ ID No. 4 in the sequence listing.

4. The use according to claim 2 or 3, characterized in that: The substance for detecting the melon flower sex molecular marker also includes restriction endonuclease SacI.

5. A method for detecting the sex characteristics of melon flowers, comprising: The method comprises the following steps: using genomic DNA of a test melon as a template, performing PCR amplification using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence list, digesting the obtained PCR products with restriction endonuclease SacI, and detecting the sizes of the digestion products. A homozygous melon having two DNA fragments of 29 bp and 166 bp is or is a candidate for a melon having male flowers on the main branch and bisexual flowers on the lower nodes of the side branches; a homozygous melon having one DNA fragment of 195 bp is or is a candidate for a melon having bisexual flowers on both the main branch and the side branches; and a heterozygous melon having three DNA fragments of 29, 166 and 195 bp is or is a candidate for a melon having male flowers on the main branch and bisexual flowers on the lower nodes of the side branches.

6. A method for breeding muskmelons with fully bloomed bisexual flowers on the main and side branches, including: The genomic DNA of the test melon was used as a template, and PCR amplification was performed using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing. The resulting PCR product was digested with the restriction endonuclease SacI, and the test melon whose digestion product was a 195 bp DNA fragment was selected as the parent for breeding.

7. A method for producing muskmelons with fully blossoming bisexual flowers on main and side branches, comprising: The genomic DNA of the test melon was used as a template, and PCR amplification was performed using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence list. The obtained PCR product was digested with the restriction endonuclease SacI, and the test melon whose digestion product was a 195 bp DNA fragment was selected for production melon.

8. Use of the substance for detecting the molecular marker of melon flower sex according to any one of claims 1 to 4 in the preparation of a product for identifying the sex trait of melon flowers; Or, use of the substance for detecting the melon flower sex molecular marker according to any one of claims 1 to 4 in melon flower sex breeding; The flower sex is as follows: the main branches have male flowers, the side branches have bisexual flowers at low nodes, and the main branches and side branches all have bisexual flowers; in, Male flowers bloom on the main branches and bisexual flowers bloom on the lower nodes of the side branches means that bisexual flowers bloom on the 1st to 3rd nodes from the base of the side branches, and male flowers bloom on the 4th node and above.

9. Use of the melon flower sex molecular marker of claim 1 in identifying the sex traits of melon flowers; Or, use of the melon flower sex molecular marker of claim 1 in breeding melon bisexual flowers; The flower sex is as follows: the main branches have male flowers, the side branches have bisexual flowers at low nodes, and the main branches and side branches all have bisexual flowers; in, Male flowers bloom on the main branches and bisexual flowers bloom on the lower nodes of the side branches means that bisexual flowers bloom on the 1st to 3rd nodes from the base of the side branches, and male flowers bloom on the 4th node and above.

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