Molecular marker co-segregated with cucumber lateral-branch-free gene and application of molecular marker
By providing the molecular marker SV-nlb, which co-segregates the cucumber no-lateral-branching gene, and using PCR amplification and electrophoresis analysis, the problem of rapid identification of the no-lateral-branching trait in cucumbers at the seedling stage was solved, realizing a rapid and accurate breeding process, which is applicable to the screening of no-lateral-branching cucumber varieties worldwide.
Patent Information
- Application Number
- CN202511280108.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2026-01-13
AI Technical Summary
In existing technologies, it is difficult to quickly and accurately select for the non-lateral branching trait during the seedling stage in cucumber breeding, resulting in a slow breeding process that is limited by environmental conditions.
A molecular marker SV-nlb, co-segregated with the cucumber non-lateral branching gene, is provided. It can be rapidly identified for cucumber seedling traits by PCR amplification using a specific primer set and combined with agarose gel electrophoresis analysis.
It enables efficient and accurate identification of the presence or absence of lateral branches during the cucumber seedling stage, shortens breeding time, is applicable to cucumber variety screening worldwide, and improves breeding efficiency.
Smart Images
Figure HSA0000301561930000011
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular genetics and breeding, specifically involving a molecular marker that co-segregates with the cucumber non-lateral branching gene and its application. Background Technology
[0002] Cucumber (Cucumis sativus L.) 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, also known as lateral branching (LB), is ubiquitous in plant growth. Its number, arrangement, and orientation influence the spatial distribution of the above-ground parts of the plant, making it an important component of plant architecture. Plant branching is closely related to its adaptability, competitive ability, and yield. In 1997, Serquen et al. located four QTL loci for multiple lateral-branching (MLB) in cucumber by constructing a genetic map. In 2003, Fazio et al. located 13 QTL loci for MLB. In 2004, Li Xiaozun et al. constructed a RAPD linkage map for cucumber, locating lateral branching and all-female traits separately, and were among the first to use forward genetics to study lateral branching genes. In the same year, Wang Gang and Pan Junsong et al. constructed a SRAP genetic linkage map for cucumber using the same parents, performing QTL mapping for the number of lateral branches and the first flowering node separately, and were among the first to study lateral branches and the first flowering node of female flowers together. In 2008, Jiang Su et al. located four QTL loci related to lateral branches using recombinant inbred lines and preliminarily revealed the regulation of lateral branch development by the F locus, linking the development of female flowers and lateral branches and other lateral tissues. In 2013, Ren Guoliang et al. studied the cucumber non-lateral branch gene nlb using materials with and without lateral branches, and located it on chromosome 1.
[0004] With the continuous expansion of greenhouse cucumber cultivation in China and the increasing market demand for processing cucumbers, different breeding requirements have been placed on the number of lateral branches on cucumber plants. Under greenhouse cultivation, excessive lateral branches alter the plant's nutrient distribution, reduce the fruit set rate of the main vine, and increase branch density, leading to poor ventilation and light penetration. Furthermore, manual pruning and pinching consume a significant amount of labor. Cucumber varieties with few or no lateral branches avoid these problems. Therefore, it is essential to breed cucumber varieties with suitable plant height, reasonable plant type, and suitability for different cultivation needs. 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 that co-segregates with the cucumber non-lateral branching gene and its application, which can be easily, quickly, and with high throughput applied to cucumber breeding practices.
[0006] This invention is achieved by providing a molecular marker co-segregating with a cucumber non-lateral branching gene. The molecular marker, SV-nlb, comprises a nucleotide sequence of 565 bp as shown in SEQ ID NO.1 and a nucleotide sequence of 27179 bp as shown in SEQ ID NO.2. The nucleotide sequence of SEQ ID NO.1 co-segregates with a cucumber non-lateral branching gene, and the nucleotide sequence of SEQ ID NO.2 co-segregates with a cucumber lateral branching gene. A primer set for amplifying the molecular marker co-segregating with the cucumber non-lateral branching gene is provided, comprising:
[0007] SV5F: 5'-GTTATCTATCACTAGAGTGGCTT-3';
[0008] SV5R: 5'-ATTGACTACATCATTTGAACCAG-3'.
[0009] A kit for determining whether cucumbers have lateral branching traits is provided, comprising the aforementioned primer set.
[0010] The application of the aforementioned molecular marker cosegregating with the cucumber non-lateral branching gene is provided for molecular marker-assisted breeding of cucumber.
[0011] Preferably, the molecular marker is used to identify whether cucumbers have lateral branching traits during the cucumber seedling stage.
[0012] A method for determining whether a cucumber has lateral branching characteristics is provided, including the following steps:
[0013] (1) Extract genomic DNA from cucumber seedling samples to be tested;
[0014] (2) Using the genomic DNA extracted in step (1) as a template, the genomic DNA is amplified using the primer set described above;
[0015] (3) The amplification products were separated by agarose gel electrophoresis;
[0016] (4) The judgment is made based on the electrophoresis results of step (3), and the specific criteria are as follows:
[0017] If the electrophoresis result of the amplification product is no amplification band, the cucumber to be tested has lateral branches; if the electrophoresis result of the amplification product is amplification band, the cucumber to be tested has no lateral branches.
[0018] 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 a total reaction system of 10 μl.
[0019] 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.
[0020] Compared with the prior art, the advantages of the present invention are as follows:
[0021] This invention provides a molecular marker that co-segregates with the cucumber's non-lateral branching gene and its application. Traditional breeding methods select based on plant morphology. Since the presence or absence of lateral branches in cucumbers can only be determined when the plant develops 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 cucumbers, accelerating the process of cucumber quality breeding. Furthermore, the molecular marker of this invention co-segregates with the cucumber's non-lateral branching trait, and all cucumber varieties from around the world can be screened for non-lateral branching / lateral branching varieties using this molecular marker. Attached Figure Description
[0022] Figure 1 The results are PCR amplification results for the molecular marker SV-nlb (M represents Marker DL2000, 9930 is the cucumber parent with lateral branches; S419 is the cucumber parent without lateral branches; F2 population represents plants randomly selected from the F2 population). Detailed Implementation
[0023] The present invention is 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, unless otherwise specified, are generally performed under conventional conditions.
[0024] Part 1: Construction of genetic segregating populations and identification of the cucumber no-lateral-branch gene
[0025] 1. Construction of the F2 segregating population
[0026] The variety with lateral branches used to construct the F2 population was 9930. The variety without lateral branches was S419. In this example, hybridization combinations were prepared using these two parents to obtain the F1 generation. The standard for the phenotype of no lateral branches was defined as lateral branches less than 1 cm. All F1 plants were single plants without lateral branches. Self-pollination of the F1 generation produced the F2 segregating population. Segregation of the lateral branching and non-lateral branching traits was observed in the F2 segregating population. The phenotypes of lateral branches / non-lateral branches were statistically analyzed, and the segregation ratio of lateral branches to non-lateral branches in the F2 segregating population was analyzed. Chi-square analysis was used to verify that the segregation ratio was 3:1. Finally, it was concluded that the trait of no lateral branches in cucumber is a dominant trait controlled by a single nuclear gene.
[0027] 2. Screening of tightly linked SV markers for the nlb gene
[0028] 2.1 Extraction of cucumber genomic DNA
[0029] 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 2mL centrifuge tube, two steel balls were added, and 750µL of CTAB extraction buffer (preheated to 60℃) was added. The tube was then ground at 60Hz for 90s and incubated at 60℃ for 0.5–1h, 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 rpm for 10 min, and 500µL 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. 10µg / mL of RNase was added. The purity was assessed by 1% agarose gel electrophoresis, and the concentration was determined using a nucleic acid analyzer. The final concentration was diluted to 50ng / µL and stored at -20℃ for later use.
[0030] 2.2 Establishment of gene pools with / without lateral branches
[0031] Ten plants with lateral branches and ten plants without lateral branches were randomly selected from the F2 segregating population to establish gene pools for cucumbers with and without lateral branches.
[0032] 2.3 Screening of Polymorphic Markers
[0033] Two parents and two gene pools were screened using 551 SSR primer pairs. The reaction mixture consisted of 30 ng genomic DNA, 0.5 μmol / L primers, 200 μmol / L dNTPs, 2 mmol / L MgCl2, 1 μl 10× PCR reactions 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.
[0034] 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.
[0035] 2.4 Fine mapping of the cucumber nlb gene
[0036] Polymorphic marker screening revealed only one marker with inter-pool polymorphism on chromosome 1. Therefore, based on genome resequencing results, multiple polymorphic InDel markers were developed on chromosome 1 of both parents (9930 and S419) to analyze their polymorphism in pools with and without lateral branches. The results showed that two InDel markers were polymorphic between the two pools, and were close to previously identified markers, indicating that the cucumber gene for lack of lateral branches is located on the anterior arm of chromosome 1. Combined with parental resequencing data, a significant chromosomal structural variation was found in the localization region between the two parents: a 27,699-base repeat. Therefore, a pair of molecular markers named SV-nlb were developed to investigate their linkage with the cucumber lateral branch phenotype.
[0037] The reaction system consisted of 30 ng of genomic DNA, 0.5 μmol / L primers, 200 μmol / L dNTPs, 2 mmol / L MgCl2, 1 μl of 10×PCR reactions 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. The amplification products were separated by 1% agarose gel electrophoresis using 1×TAE buffer at a constant power of 120 W for 15 min.
[0038] refer to Figure 1The 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 SV-nlb marker was the banding pattern of S419 without lateral branches, the phenotype of a single plant was without lateral branches; when the banding pattern of the SV-nlb marker was the banding pattern of 9930 with lateral branches, there was no obvious band, and the phenotype of a single plant was with lateral branches. Finally, it was found that the SV-nlb marker was completely linked to the cucumber gene nlb that prevents lateral branches.
Claims
1. A molecular marker co-segregating with the cucumber non-lateral branching gene, characterized in that, The molecular marker is SV-nlb, which includes a nucleotide sequence of 565 bp as shown in SEQ ID NO.1 and a nucleotide sequence of 27179 bp as shown in SEQ ID NO.
2. The nucleotide sequence of SEQ ID NO.1 co-segregates with the cucumber non-lateral branching gene, and the nucleotide sequence of SEQ ID NO.2 co-segregates with the cucumber lateral branching gene.
2. The primer pair for amplifying the molecular marker co-segregating with the cucumber non-lateral branching gene as described in claim 1, characterized in that, include: SV5F: 5'-GTTATCTATCACTAGAGTGGCTT-3'; SV5R: 5'-ATTGACTACATCATTTGAACCAG-3'.
3. A reagent kit for determining whether cucumbers have lateral branching characteristics, characterized in that, It includes the primer set as described in claim 2.
4. The application of the molecular marker co-segregating with the cucumber non-lateral branching gene as described in claim 1, characterized in that, Molecular marker-assisted breeding for cucumbers.
5. The application according to claim 4, characterized in that, The molecular markers are used to identify whether cucumbers have lateral branching traits during the seedling stage.
6. A method for determining whether a cucumber has lateral branching characteristics, characterized in that, Includes 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 in claim 2; (3) The amplification products were separated by agarose 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 no amplification band, the cucumber to be tested has lateral branches; if the electrophoresis result of the amplification product is amplification band, the cucumber to be tested has no lateral branches.
7. The method according to claim 6, characterized in that, 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 a total reaction system of 10 μl.
8. The method according to claim 6, characterized in that, The amplification program in step (2) is as follows: 94℃ for 5 min; 32 cycles; 94℃ for 30 s; 57℃ for 30 s; 72℃ for 30 s; 72℃ for 5 min.