Molecular marker and reagent for identifying all-bisexual floral character of muskmelon

Abstract

The invention discloses a molecular marker and a reagent for identifying all-bisexual floral character of muskmelon. The muskmelon flower gender molecular marker is found to be related to the gender of muskmelon flowers, and the muskmelon flower gender molecular marker is the 30th nucleotide corresponding to SEQ ID No.1 in a sequence table in a muskmelon genome, and is C or T. Experiments prove that the muskmelon flower gender molecular marker can be used for identifying the character of muskmelon flowers, the muskmelon flower gender molecular marker can be used for molecular marker-assisted breeding, and the genotype of all-bisexual flowers can be selected for muskmelon production. The method has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and is used for identifying the traits of the pan-hermaphroditic floral system of melon and a molecular marker and a reagent. Background Art

[0002] Muskmelon is widely cultivated all over the world. It is the earliest melon grown as a fruit in China and is also an important horticultural crop with great economic value.

[0003] At present, most commercially cultivated melon varieties are monoecious with male flowers, with male flowers on the main branches and bisexual flowers on the lower nodes of the side branches. The fruits of melons with male flowers are developed from bisexual flowers, and the number of bisexual flowers on the side branches is limited. After the melons bloom, they are often affected by pollination, nutritional status and other conditions, which limits the melon yield. Therefore, improving the new planting model with all-hermaphroditic flower lines as the main cultivated plants, adopting mixed planting with all-male plants, and using bumblebee pollination is of great significance to improve the fruit setting rate of melons, increase yields, and reduce the cost of melon cultivation. The acquisition and identification of melon all-hermaphroditic flower lines is of great significance.

[0004] Using molecular markers that are closely linked to target genes to carry out molecular marker-assisted selection breeding can improve the efficiency and accuracy of the selection of target traits, accelerate the breeding process, and broaden the application scope of target genes, which is a necessary condition for molecular marker-assisted selection breeding. Therefore, the development of molecular markers that are closely linked to the pan-hermaphroditic flower trait has important practical value for improving the efficiency of breeding pan-hermaphroditic flower parents, expanding the application scope of pan-hermaphroditic flower genes, and promoting the stable development of the melon industry. Summary of the invention

[0005] The technical problem to be solved by the invention is how to identify the sex traits of melon flowers.

[0006] In order to solve the above technical problems, the present invention first provides the use of melon flower sex molecular markers or substances for detecting the melon flower sex molecular markers in identifying or assisting in identifying melon flower sex traits;

[0007] 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.

[0008] In the above application, the substance for detecting the melon flower sex molecular marker may include a primer pair that can amplify the melon flower sex molecular marker, the primer pair is composed of single-stranded DNA named P1 and P2, P1 is a single-stranded DNA that specifically binds to the 30th upstream of SEQ ID No.1 in the melon genome, and P2 is a single-stranded DNA that specifically binds to the 30th downstream of SEQ ID No.1 in the melon genome.

[0009] In the above application, the sequence of P1 may be SEQ ID No. 3 in the sequence list; the sequence of P2 may be SEQ ID No. 4 in the sequence list.

[0010] In the above application, the substance for detecting the melon flower sex molecular marker may also include restriction endonuclease SacI.

[0011] In the above application, the substance for detecting the melon flower sex molecular marker can be the primer pair, or can be composed of the primer pair and restriction endonuclease SacI.

[0012] The present invention also provides a method for detecting the sex trait of melon flowers. The method comprises: using the genomic DNA of the melon to be tested 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 product using restriction endonuclease SacI, detecting the size of the digestion product, a homozygous melon whose digestion product is 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 side branches at lower nodes, a homozygous melon whose digestion product is one DNA fragment of 195 bp is or is a candidate for a melon with bisexual flowers on the main branch and side branches, and a heterozygous melon whose digestion products are 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 side branches at lower nodes.

[0013] Among them, melons with male flowers on the main branches and bisexual flowers on the lower nodes of the side branches refer to melons with bisexual flowers on the 1st to 3rd nodes from the base of the side branches, and male flowers on the 4th node and above.

[0014] The present invention also provides a melon breeding method, which comprises: using the genomic DNA of the melon to be tested 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 table, digesting the obtained PCR product with a restriction endonuclease SacI, and selecting the melon to be tested whose digestion product is a DNA fragment of 195 bp as a parent for breeding.

[0015] The present invention also provides a method for producing melons, which comprises: using the genomic DNA of the melon to be tested 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 table, digesting the obtained PCR product with a restriction endonuclease SacI, and selecting the melon to be tested whose digestion product is a DNA fragment of 195 bp to produce the melon.

[0016] In the above, the reaction system for PCR amplification using the primer pair composed of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence table can be: 7 μL of 2×GoTaq Mix (promega, M712B), 1 μL of P1, 1 μL of P2, and 20 μL of genomic DNA of the melon to be tested supplemented with sterile ultrapure water. The concentrations of P1 and P2 in the system are both 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 table can be: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 15 seconds, 35 cycles, and extension at 72°C for 5 minutes.

[0018] The substance for detecting the melon flower sex molecular marker also falls within the protection scope of the present invention.

[0019] The use of the substance for detecting the melon flower sex molecular marker in the preparation of a product for identifying or assisting in identifying the sex trait of a melon flower also falls within the protection scope of the present invention.

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

[0021] The application of the melon flower sex molecular marker in identifying or assisting in identifying the sex traits of melon flowers also falls within the protection scope of the present invention.

[0022] The application of the melon flower sex molecular marker in melon breeding also falls within the protection scope of the present invention.

[0023] In the present invention, the melon flower traits are all traits of flowers grown naturally.

[0024] Experiments have shown that the melon flower sex molecular marker of the present invention can be used to identify the traits of melon flowers, and the melon flower sex molecular marker of the present invention can be used for molecular marker-assisted breeding, and the genotype of fully bisexual flowers can be selected for melon production. The present invention has a good application prospect.

[0025] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0026] Description of biological material deposit

[0027] Abbreviation of depository unit: CGMCC

[0028] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0029] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0030] Date of deposit: May 8, 2023

[0031] CGMCC Registration Number: CGMCC No.26394

[0032] Classification: Cucumis melo

[0033] Plant number: 13C-WT

[0034] Description of biological material deposit

[0035] Abbreviation of depository unit: CGMCC

[0036] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0037] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0038] Date of deposit: May 8, 2023

[0039] CGMCC Registration Number: CGMCC No.26395

[0040] Classification: Cucumis melo

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

[0042] Figure 1 The first lane on the left is the DNA molecular weight standard. DETAILED DESCRIPTION

[0043] The experimental methods in the following examples, unless otherwise specified, 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. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were set up for three repeated experiments, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position 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 Huaisong (Beijing, China), and its main branches have male flowers and the side branches have bisexual flowers at the lower nodes (i.e., bisexual flowers are bloomed at the 1st to 3rd nodes from the base of the side branches, and male flowers are bloomed at the 4th node and above). 13C-WT has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 8, 2023, with the deposit number CGMCC No. 26394.

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

[0046] Example 1: A set of reagents for detecting the sex characteristics of melon flowers can identify the characteristics of melon flowers with full hermaphroditic flowers

[0047] This embodiment provides a molecular marker related to the sex of melon flowers, which is the 30th nucleotide of SEQ ID No.1 in the sequence list, recorded as the 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 the melon is defined according to whether the melon flower sex molecular marker in the genomic DNA is C or T. The melon has two homologous chromosomes corresponding to SEQ ID No.1 and both are SEQ ID No.1, which is the AA genotype; the melon has two homologous chromosomes corresponding to SEQ ID No.1, one is SEQ ID No.1 and the other is SEQ ID No.2, which is the Aa genotype; the melon has two homologous chromosomes corresponding to SEQ ID No.1 and both are SEQ ID No.2, which is the aa genotype. Among them, the AA genotype and the aa genotype can be stably inherited.

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

[0050] P1: 5′-ATTATAATCCTTCTTCTTCTTCTC G AG CT-3′ (SEQ ID No. 3 in the sequence list, the underlined part is the mutant nucleotide introduced by the 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 variety were selected for the experiment:

[0054] P1: melon 13C-WT;

[0055] P2:13C-68;

[0056] F1: the first generation of the cross between melon 13C-WT and 13C-68;

[0057] F2: The second generation of hybridization between melon 13C-WT and 13C-68 (F2, i.e., the self-pollination generation of F1).

[0058] 2. Identification of the traits of melon's fully bisexual flowers

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

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

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

[0062] The sex of the flowers of each plant was observed. The results showed that the melons with genotypes AA and Aa had male flowers on the main branches and bisexual flowers on the lower nodes of the side branches, while the melons with genotype aa had bisexual flowers on the main branches and side branches. Some of the results are shown in Table 1.

[0063] Table 1. Genotypes and phenotypes of the melons tested

[0064]

[0065]

[0066] In Table 1, bisexual flowers blooming at low nodes of lateral branches refers to bisexual flowers blooming at the 1st to 3rd nodes from the base of the lateral branches, and male flowers blooming at the 4th node and above.

[0067] It is explained that the melon flower sex molecular marker and the set of reagents for identifying or assisting in identifying the sex traits of melon flowers of the present invention can identify the sex of melon flowers and can be used for molecular marker-assisted breeding.

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

[0069] SEQ ID No.1:

[0070]

[0071] SEQ ID No.2:

[0072]

[0073] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of melon flower sex molecular markers or substances for detecting the melon flower sex molecular markers in identifying or assisting in identifying melon flower sex traits; 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.

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, the primer pair is composed of single-stranded DNA named P1 and P2, the P1 is a single-stranded DNA that specifically binds to the 30th upstream of SEQ ID No.1 in the melon genome, and the P2 is a single-stranded DNA that specifically binds to the 30th downstream of SEQ ID No.1 in the melon genome.

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 a melon flower, comprising: The genomic DNA of the tested melon is used as a template, and a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence list is used for PCR amplification. The obtained PCR product is digested with restriction endonuclease SacI, and the size of the digested product is detected. The homozygous melon whose digested products are 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 lower nodes of the side branches, the homozygous melon whose digested product is one DNA fragment of 195 bp is or is a candidate for a melon with bisexual flowers on the main branch and all the side branches, and the heterozygous melon whose digested products are 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 lower nodes of the side branches.

6. A method for breeding melons, comprising: The genomic DNA of the melon to be tested was used as a template, and a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence table was used for PCR amplification. The obtained PCR product was digested with restriction endonuclease SacI, and the melon to be tested whose digestion product was a DNA fragment of 195 bp was selected as a parent for breeding.

7. A method for producing melons, comprising: The genomic DNA of the melon to be tested is used as a template, and a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence table is used for PCR amplification. The obtained PCR product is digested with restriction endonuclease SacI, and the melon to be tested whose digestion product is a DNA fragment of 195 bp is selected for production melon.

8. The substance for detecting the molecular marker of melon flower sex as described in any one of claims 1-4.

9. Use of the substance for detecting the melon flower sex molecular marker according to any one of claims 1 to 4 in preparing a product for identifying or assisting in identifying the sex trait of melon flowers; Or, use of the substance for detecting the melon flower sex molecular marker as described in any one of claims 1-4 in melon breeding.

10. Use of the melon flower sex molecular marker of claim 1 in identifying or assisting in identifying the sex trait of melon flowers; Or, use of the melon flower sex molecular marker described in claim 1 in melon breeding.

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