Molecular marker closely linked to cucumber short- internode gene and its application

Abstract

The application belongs to the field of molecular genetics breeding, and particularly relates to a molecular marker closely linked to a cucumber short lateral branch gene and application, the molecular marker is Indel-01, includes a nucleotide sequence shown in SEQ ID NO.1 with a length of 85bp, a nucleotide sequence shown in SEQ ID NO.2 with a length of 82bp, the nucleotide sequence shown in SEQ ID NO.1 is closely linked to the cucumber short lateral branch gene, and the nucleotide sequence shown in SEQ ID NO.2 is closely linked to the cucumber multi-lateral branch gene. The molecular marker of the application is closely linked to the cucumber short lateral branch trait, and can be used for cucumber molecular marker assisted breeding, and accelerates the process of cucumber quality breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and breeding, specifically involving a molecular marker closely linked to the cucumber short lateral branch gene and its application. Background Technology

[0002] cucumber( Cucumissativus Cucumber (Cucumis spp.) is an annual vine or climbing herbaceous plant belonging to the genus Cucumis in the family Cucurbitaceae. As one of the world's ten most important vegetable crops, cucumber is also one of the main vegetable crops cultivated in my country. The main cucumber varieties cultivated in my country are vine-type varieties.

[0003] Branching, as a key agronomic trait, directly impacts plant architecture and crop yield. In flowering plants, lateral bud development originates from the formation of axillary meristem (AM) in the leaf axils. This meristem further differentiates into axillary buds, containing a small number of leaf primordia and meristems, at which stage they either enter dormancy or continue to grow, forming branches. Excessive branching often competes for nutrients and light resources, thus negatively affecting crop yield. In fact, suppressing branching has been an important selection trait during crop domestication. Cucumber (Cucumis sativus L.), a globally widely cultivated vegetable crop, can be eaten fresh or processed into pickles. It was domesticated from its highly branching wild ancestor, Cucumis sativus var. hardwickii. Although lateral branching in cultivated cucumbers is significantly reduced compared to hardwickii, the degree of branching suppression still varies depending on the variety, growing region, and market type. In modern cucumber production, especially for the fresh food market, it is often necessary to manually remove excess branches. This process is not only time-consuming and labor-intensive, but also increases production costs. Therefore, a thorough analysis of the regulatory mechanisms of cucumber lateral branch development is of great value for promoting cucumber breeding and production practices.

[0004] Currently, the cultivation area of ​​greenhouse cucumbers in China is continuously increasing, and the market demand for processing cucumbers is becoming increasingly prominent. This poses new requirements for the breeding direction of cucumber lateral branches. Multiple lateral branches under greenhouse conditions not only disrupt nutrient supply and inhibit fruit setting on the main vine, but also easily lead to poor ventilation and light penetration within the plant canopy, and require extensive manual pruning. Varieties with few or no lateral branches can effectively overcome these bottlenecks. Therefore, breeding cucumber varieties with suitable plant height, optimized plant type, and the ability to flexibly adapt to different cultivation needs is an inevitable choice for the industry's development. Marker-assisted selection using molecular markers closely linked to the target trait is a highly effective method in cucumber genetic breeding. Molecular markers select for target traits at the DNA level, offering advantages such as high efficiency, speed, and independence from environmental conditions. Selection can be performed during the seedling stage, accelerating the breeding process. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a molecular marker and its application that is closely linked to the cucumber short lateral branch gene, which can be easily, quickly, and with high throughput applied to cucumber breeding practices.

[0006] The purpose of this invention is to meet the requirements of selecting suitable plant types under different cultivation conditions by providing an Indel molecular marker tightly linked to the cucumber short lateral branch gene. This marker can clearly show differences using polyacrylamide gel electrophoresis, facilitating its application in molecular-assisted breeding of short lateral branches. This paper uses molecular markers, BSA (Bulked Segregant Analysis), and sequencing between parents to discover that a 3bp deletion in the short lateral branch gene in multi-lateral branching parents leads to the loss of gene function, ultimately resulting in the multi-lateral branching phenotype. Using insertion / deletion mutations, an Indel molecular marker tightly linked to the cucumber short lateral branch gene was developed. This molecular marker, characterized by its tight linkage to the short lateral branch gene, provides a molecular marker tightly linked to the cucumber short lateral branch gene. The molecular marker of this invention can be easily, rapidly, and with high throughput applied in breeding practices.

[0007] This invention is achieved by providing a molecular marker tightly linked to a cucumber short lateral branch gene. The molecular marker comprises an 85 bp nucleotide sequence as shown in SEQ ID NO.1 and an 82 bp nucleotide sequence as shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.1 is tightly linked to the cucumber short lateral branch gene, and the nucleotide sequence shown in SEQ ID NO.2 is tightly linked to a cucumber multi-lateral branch gene. A primer set for amplifying the molecular marker tightly linked to the cucumber short lateral branch gene is provided, comprising: Indel-01F: 5'-TCAAACTGGGAAGTGATTGGATT-3'; Indel-01R: 5'-AAACTACTGGTGGAGAGGAATCA-3'.

[0008] A kit for determining whether cucumbers have short lateral branching traits is provided, comprising the aforementioned primer set.

[0009] The application of the molecular markers closely linked to the cucumber short lateral branch gene is provided for molecular marker-assisted breeding of cucumber.

[0010] Preferably, the molecular marker is used to identify the short lateral branch trait of cucumber during the cucumber seedling stage.

[0011] A method for determining the short lateral branch trait in cucumber is provided, comprising the following steps: (1) Extract genomic DNA from cucumber seedling samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, the genomic DNA is amplified using the primer set described above; (3) The amplification products were separated by polyacrylamide gel electrophoresis; (4) The judgment is made based on the electrophoresis results of step (3), and the specific criteria are as follows: If the electrophoresis result of the amplification product is 9930 bands, the cucumber to be tested has short lateral branches; if the electrophoresis result of the amplification product is M12 bands, the cucumber to be tested has multiple lateral branches.

[0012] Preferably, the amplification reaction system in step (2) is as follows: 30 ng genomic DNA, 0.5 µmol / L primers, 200 µmol / L dNTPs, 2 mmol / L MgCl2, 1 μl 10×PCR reactions buffer, 0.6 U Taq DNA polymerase, and the total reaction system is 10 µl.

[0013] Preferably, the amplification program in step (2) is: 94℃ for 5 min; 32 cycles; 94℃ for 30 s; 57℃ for 30 s; 72℃ for 30 s; 72℃ for 5 min.

[0014] Compared with the prior art, the advantages of the present invention are as follows: This invention provides a molecular marker closely linked to the short lateral branch gene in cucumber and its application. Traditional breeding methods select based on plant morphology. Since the trait of long and short lateral branches in cucumber can only be determined when the plant has lateral branches, it is time-consuming. The molecular marker of this invention can be identified in the cucumber seedling stage, which is both time-saving and accurate. It can be used for molecular marker-assisted breeding of cucumber, accelerating the process of cucumber quality breeding. Furthermore, the molecular marker of this invention is closely linked to the short lateral branch gene in cucumber, and all cucumber varieties from around the world can be screened for short / multiple lateral branch varieties using this molecular marker. Attached Figure Description

[0015] Figure 1 The results are PCR amplification results for molecular markers (M represents Marker DL500, 9930 is the cucumber short lateral branch parent; M12 is the cucumber multi-lateral branch parent; F2 population represents plants randomly selected from the F2 population). Detailed Implementation

[0016] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0017] Part 1: Construction of genetic segregating populations and identification of the short lateral branch gene in cucumber 1. Construction of the F2 segregating population The short-lateral-branch variety used to construct the F2 population was 9930. The multi-lateral-branch variety was M12. In this example, these two parents were used to create a hybrid combination, yielding the F1 generation. The short-lateral-branch phenotype was defined as lateral branches less than 1 cm in length, and all F1 plants were single plants with short lateral branches. Self-pollination of the F1 generation produced the F2 segregating population. Segregation of short and multi-lateral-branch traits was observed in the F2 segregating population. The short / multi-lateral-branch phenotype was statistically analyzed, and the segregation ratio of short to multi-lateral branches in the F2 segregating population was verified using chi-square analysis, confirming a 3:1 segregation ratio. Finally, it was concluded that the short-lateral-branch trait in cucumber is a dominant trait controlled by a single nuclear gene.

[0018] 2. Screening for markers closely linked to short lateral branch genes 2.1 Extraction of cucumber genomic DNA Total DNA was extracted from leaves of the parental plants and F1 and F2 isolates using the CTAB method. The method was as follows: A single young leaf was placed in a 2 mL centrifuge tube, two steel balls were added, and 750 μL of CTAB extraction buffer (preheated to 60℃) was added. The tube was then ground at 60 Hz for 90 s and incubated at 60℃ for 0.5–1 h, with gentle shaking once during incubation. An equal volume of chloroform was added, and the tube was gently shaken for 5 min. The tube was centrifuged at 12000 r / min for 10 min, and 500 μL of the supernatant was collected. Two volumes of anhydrous ethanol were added, and the tube was incubated at -20℃ for 30 min. The tube was centrifuged again to remove the supernatant, and washed twice with 70% ethanol. The tube was then air-dried and dissolved in 1×TE buffer. 10 μg / mL of RNase was added. Purity was assessed by 1% agarose gel electrophoresis, and concentration was determined using a nucleic acid analyzer. The final concentration was diluted to 50 ng / μL and stored at -20℃ for later use.

[0019] 2.2 Establishment of short / multiple lateral branch gene pools Ten individuals with short lateral branches and ten individuals with multiple lateral branches were randomly selected from the F2 segregating population to establish gene pools for cucumbers with short lateral branches and cucumbers with multiple lateral branches.

[0020] 2.3 Screening of Polymorphic Markers Two parents and two gene pools were screened using a molecular marker developed in this invention. The reaction system consisted of 30 ng genomic DNA, 0.5 µmol / L primers, 200 µmol / L dNTPs, 2 mmol / L MgCl2, 1 μl 10×PCR reaction buffer, and 0.6 U Taq DNA polymerase, with a total reaction volume of 10 µl. The PCR amplification program was as follows: 94℃ for 5 min; 32 cycles; 94℃ for 30 s; 57℃ for 30 s; 72℃ for 30 s; 72℃ for 5 min.

[0021] The amplified products were separated by 8% denaturing polyacrylamide gel electrophoresis (TBE) with a buffer of 1×TBE for 1.5-2 hours. After electrophoresis, silver staining was performed. The staining method was as follows: the gel was placed in a staining solution (2 g / L silver nitrate) and shaken for 15 minutes. After staining, the gel was rinsed in ultrapure water and then placed in a plastic container containing the developing solution. The solution was gently shaken until the bands were clear, then rinsed in tap water. The results were analyzed and photographed. The developing solution was prepared by mixing 30 g NaOH and 4 ml formaldehyde in 1 L of distilled water.

[0022] refer to Figure 1 The results showed that the banding pattern of the molecular marker was completely consistent with the phenotype of the plant. That is, when the banding pattern of the marker was the short lateral branch 9930, the phenotype of the single plant was short lateral branches; when the banding pattern of the marker was the multi-lateral branch M12, the phenotype of the single plant was multi-lateral branches. Finally, it was found that the Indel-01 marker was closely linked to the cucumber short lateral branch gene.

Claims

1. The application of primer pairs of molecular markers closely linked to the cucumber short lateral branch gene in identifying the cucumber short lateral branch trait during the cucumber seedling stage, characterized in that, The molecular marker is Indel-01, and its nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2. The molecular marker Indel-01, as shown in SEQ ID NO.1, is tightly linked to the cucumber short lateral branch gene, and the molecular marker Indel-01, as shown in SEQ ID NO.2, is tightly linked to the cucumber multi-lateral branch gene. The primer pair includes: Indel-01F: 5'-TCAAACTGGGAAGTGATTGGATT-3'; Indel-01R: 5'-AAACTACTGGTGGAGAGGAATCA-3'; The cucumbers in question are cucumber varieties 9930, M12, and their hybrid offspring.

2. A method for identifying the short lateral branch trait in cucumber, characterized in that, Includes the following steps: (1) Extract genomic DNA from the cucumber seedling samples to be tested, wherein the cucumbers are cucumber varieties 9930, M12 and their hybrid offspring; (2) Using the genomic DNA extracted in step (1) as a template, the genomic DNA is amplified using primer pairs of molecular markers closely linked to the cucumber short lateral branch gene. The primer pairs include: Indel-01F: 5'-TCAAACTGGGAAGTGATTGGATT-3'; Indel-01R: 5'-AAACTACTGGTGGAGAGGAATCA-3'; (3) The amplification products were separated by polyacrylamide gel electrophoresis; (4) The judgment is made based on the electrophoresis results of step (3), and the specific criteria are as follows: If the electrophoresis results of the amplified product are consistent with the electrophoresis results of cucumber variety 9930 amplified using the primer pair, then the cucumber to be tested has short lateral branches. If the electrophoresis results of the amplified product are consistent with the electrophoresis results of cucumber variety M12 amplified using the primer pair, then the cucumber to be tested has multiple lateral branches.

3. The method according to claim 2, characterized in that, The amplification program in step (2) is as follows: 94℃ for 5 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 32 cycles; 72℃ for 5 min.

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