A caps marker primer for identifying the plant height trait of brassica oleracea and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a CAPS marker primer for identifying the plant height trait of Brassica oleracea and application thereof, a nucleotide sequence of the CAPS marker primer is shown as SEQ ID NO. 6~SEQ ID NO. 7, the CAPS marker for identifying the plant height of Brassica oleracea, the CAPS marker primer and the application method provided by the application lay a molecular foundation for the breeding improvement of the plant height of Brassica oleracea.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a CAPS marker primer for identifying the plant height trait of kale and its application. Background Technology

[0002] kale ( Brassica oleracea var. acephala Kale (Brassica oleracea var. rubrum) is a biennial herbaceous foliage plant belonging to the Brassicaceae family, valued for both its ornamental and economic aspects. Its leaves exhibit diverse shapes and vibrant colors, and it demonstrates exceptional cold hardiness and resilience, making it a widely used core ornamental flower in autumn and winter landscaping. Plant height, a key indicator of the ornamental quality of kale, directly affects not only the plant's visual appeal and lodging resistance but also its application in landscaping. Rich phenotypic diversity in plant height can effectively expand the application of kale in landscaping and green space ecological configuration. Therefore, developing molecular markers for kale plant height, creating germplasm resources, and breeding varieties are important goals in kale breeding.

[0003] CAPS (Capsule-Activated Polymorphic Sequence) markers are molecular marker techniques that combine PCR amplification with restriction endonuclease digestion to detect SNP polymorphisms. They offer advantages such as co-dominant inheritance, high stability and reproducibility, and ease of operation and control. Currently, they are widely used in various plant trait studies, but their application is still limited in the study of genes related to plant height in kale. BoFBK The CAPS marker and primers have not yet been developed for application. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a CAPS marker primer for identifying the plant height trait of kale and its application.

[0005] CAPS marker primers for identifying the plant height trait of kale, the nucleotide sequences of which are shown in SEQ ID NO.6~SEQ ID NO.7.

[0006] Amplification using the primers of this invention produces a high-yield DNA amplification product containing one restriction enzyme site, which can be... Hpy The DNA was digested with 166II restriction endonuclease into 277bp and 61bp. The DNA amplification product of the dwarf variety had no restriction site and the band length was 338bp, which can be used to identify the plant height trait of kale.

[0007] The application of the CAPS marker primers in identifying the plant height trait of kale.

[0008] Preferably, the steps for identifying the plant height trait of kale are as follows: Using the DNA of the kale genome as a template, PCR amplification was performed using the CAPS-labeled primers to obtain PCR products; use Hpy The PCR product was digested with 166II enzyme, and then subjected to agarose gel electrophoresis. If a 338bp electrophoretic band is obtained, the SNP site is C, indicating a dwarf kale; if two electrophoretic bands of 277bp and 61bp are obtained, the SNP site is T, indicating a tall kale. The SNP site is position 590 of the sequence shown in SEQ ID NO.3.

[0009] Preferably, kale with the SNP site T is used to cultivate tall kale plants.

[0010] Preferably, kale with the SNP site C is used to cultivate kale with low plant height.

[0011] Preferably, the PCR amplification system is: 2×Taq enzyme buffer, DNA template, CAPS-labeled primers shown in SEQ ID NO.6, CAPS-labeled primers shown in SEQ ID NO.7, and water; The volume ratio of 2×Taq enzyme buffer, DNA template, CAPS-labeled primers shown in SEQ ID NO.6, CAPS-labeled primers shown in SEQ ID NO.7, and water is 5:1:1:1:2.

[0012] Preferably, the PCR amplification conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 7 min; storage at 4°C.

[0013] Preferably, the enzyme digestion system is: PCR product, Hpy 166II restriction endonuclease, 10× digestion buffer, water; PCR products, Hpy The volume ratio of 166II restriction endonuclease, 10× digestion buffer, and water was 4:1:1:4.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a CAPS marker for identifying the height of kale plants, CAPS marker primers, and their application methods, laying a molecular foundation for the breeding and improvement of kale plant height. Attached Figure Description

[0015] Figure 1 For the present invention BoFBK Gene cloning.

[0016] Figure 2 Developed for the CAPS590 mark of this invention; in BoFBK A CAPS marker was developed at position 590 of the CDS sequence, and the hypergenic type contains a restriction endonuclease near this position. Hpy The 166II restriction site cannot be cleaved in the dwarf variety due to a mutation from "C" to "T". Hpy Digestion with 166II restriction enzyme, restriction endonuclease Hpy The recognizable restriction site of 166II is: BoFBK The complementary chains, therefore this figure shows S0836 and 6BZ. BoFBK The partial sequence of the gene containing the restriction enzyme site is reverse-complemented and compared with the restriction enzyme site.

[0017] Figure 3 This invention presents phenotypic diagrams and enzyme digestion bands of tall and dwarf varieties of kale. A represents the phenotypic diagrams of the tall and dwarf inbred lines. The tall inbred lines include: 1: S0836, 3: Deep Flamingo, 5: Yellow Rose; the dwarf inbred lines include: 2: 6BZ, 4: P29, 6: Nagoya White. B represents the enzyme digestion bands. M: Trans2KPlusⅡ DNA Marker, 1: S0836, 2: 6BZ, 3: Deep Flamingo, 4: P29, 5: Yellow Rose, 6: Nagoya White. The tall varieties (1, 3, 5) can be digested, yielding 277bp and 61bp fragments respectively. Due to the small size of the 61bp fragment, the band is not obvious after digestion. The dwarf varieties (2, 4, 6) cannot be digested, yielding 338bp. Detailed Implementation

[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0019] This invention constructed a genetic segregating population using the tall kale inbred line 'S0836' and the dwarf kale inbred line '6BZ'. Through pooled sequencing, cloning, and functional verification, a plant height-related gene was identified. BoFBK The discovery of functional genes related to plant height provides a theoretical basis for breeding kale with plant height traits.

[0020] The methods for cultivating seedlings of kale inbred lines 'S0836' and '6BZ' are as follows; The 'S0836' was obtained by using the kale variety 'Sunrise' as the original resource and employing a continuous self-pollination method, resulting in a high-yielding inbred line 'S0836' after 7 generations of continuous self-pollination. The '6BZ' line was obtained by using the kale variety 'Kamome White' as the original resource and employing a continuous self-pollination method, resulting in a dwarf inbred line '6BZ' after seven generations of continuous self-pollination.

[0021] The location was found using map-based cloning technology. BoFBK Genes. Due to in BoFBK A SNP mutation from "C" to "T" occurred at position 590 of the gene's CDS, resulting in a dwarfing phenotype. To further investigate... BoFBK Applying genes to improve the height of kale plants requires the development of a simple and high-throughput molecular marker to predict different plant heights. In SNP variation detection techniques, CAPS markers can efficiently and rapidly detect SNP sites through simple PCR amplification, restriction enzyme digestion, and product gel electrophoresis. This has made it an important molecular marker technique in biological research, widely used in genetic mapping, germplasm identification, and assisted breeding.

[0022] Example 1 kale BoFBK Cloning of genes (1) Cultivation of kale Seedlings of the tall inbred line 'S0836' and the dwarf inbred line '6BZ' of kale were cultivated using conventional methods.

[0023] (2) Extraction of genomic DNA from kale DNA was extracted from 'S0836' and '6BZ' using the CTAB method.

[0024] (3) Primer design for cloning the BoFBK CDS sequence of the kale plant height gene: Download from the cabbage database (http: / / plants.ensembl.org / Brassica_oleracea / Info / Index) Bo6g125560 Gene sequence, and design primers for CDS sequence amplification of this gene. The primer sequences are as follows: BoFBK-F: 5'-ATGGAACTCATCCCTAATCTC-3', denoted as SEQ ID NO.1; BoFBK-R: 5'-TTAAACCTCTAAGAAGCAACC-3', denoted as SEQ ID NO.2.

[0025] (4) PCR amplification PCR reaction system: including 5 μL of 2×Taq enzyme buffer, 1 μL of DNA template, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of deionized water; PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 47°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C extension for 7 min; store at 4°C. (5) Agarose gel electrophoresis detection PCR products were detected by 1% agarose gel electrophoresis, and the bands were observed using a gel imaging system. Figure 2 ).

[0026] (6) Recovery of amplification products The agarose gel recovery kit was used for recovery. (7) The recovered product was ligated into a cloning vector, transformed into E. coli, and plasmids were extracted; (8) Sequencing: The plasmid ligated with the cloning vector was sequenced, and the full-length sequences of the parents were obtained as follows:

[0027]

[0028] Example 2 Development of CAPS molecular markers (1) Synthesize molecular markers for distinguishing plant height traits in kale Based on the nucleotide sequence obtained in Example 1, differential sites were compared using the Multalin online software (http: / / multalin.toulouse.inra.fr / multalin / ). Sequences containing differential sites were introduced with zero mismatches in dCAPSFinder2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html), and the restriction site SEQ ID NO.5 was determined to be 5'-GTN^NAC-3'. Two oligonucleotide primers, CAPS590-F and CAPS590-R, were designed and synthesized as molecular markers for identifying the plant height trait of kale.

[0029] The primer sequences for CAPS590-F and CAPS590-R are as follows: CAPS590-F: 5'-ACGACGAAGAGAAGCAAGCG-3', denoted as SEQ ID NO.6; CAPS590-R: 5'-ACGACGAAGAGAAGCAAGCG-3', denoted as SEQ ID NO.7.

[0030] In tall varieties of kale, SNP590 is C, and restriction endonucleases can be found near this site. Hpy 166Ⅱ is identified and cleaved, but in the dwarf type, this site is T and cannot be identified. Hpy 166Ⅱ identification, CAPS590 marker development at this site ( Figure 3 Theoretically, high-yield DNA amplification products contain one restriction enzyme site, which can be... Hpy The 166II restriction endonuclease digested the DNA into 277bp and 61bp fragments. The dwarf DNA amplification product had no restriction site and a band length of 338bp.

[0031] Example 3 A method for determining the plant height of kale (1) Extracting genomic DNA from kale; (2) Using the DNA extracted in step (1) as a template, PCR amplification reaction was carried out using specific primers CAPS590-F and CAPS590-R; PCR reaction system: including 5 μL of 2×Taq enzyme buffer, 1 μL of DNA template, 1 μL of CAPS590-F primer, 1 μL of CAPS590-R primer, and 2 μL of deionized water; PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 7 min; store at 4°C. Enzyme digestion reaction system: 4 μL of PCR product, Hpy 1 μL of 166II restriction endonuclease, 1 μL of 10× digestion buffer, and 4 μL of deionized water; Enzyme digestion procedure: 37℃ for 3 hours.

[0032] (3) Agarose gel electrophoresis detection The PCR products were detected by 2% agarose gel electrophoresis. The positions of the bands were observed using a gel imaging system and the images were saved. If there were two specific bands of 277bp and 61bp, it was a tall kale; if there was one specific band of 338bp, it was a dwarf kale.

[0033] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0034] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0035] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. CAPS marker primers for identifying the plant height trait of kale, characterized in that, The nucleotide sequences of the CAPS-labeled primers are shown in SEQ ID NO.6~SEQ ID NO.

7.

2. The application of the CAPS-labeled primers according to claim 1 in identifying the plant height trait of kale, characterized in that, The identification of kale is based on the electrophoresis results. If a 338bp band is obtained, the SNP site is C, indicating that it is a dwarf kale. If two bands of 277bp and 61bp are obtained, the SNP site is T, indicating that it is a tall kale. The SNP site is position 590 of the sequence shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, The steps for identifying the plant height trait of kale are as follows: Using the DNA of the kale genome as a template, PCR amplification was performed using the CAPS-labeled primers to obtain PCR products; use Hpy The PCR product was digested with 166II enzyme, and then subjected to agarose gel electrophoresis.

4. The application according to claim 3, characterized in that, Kale with the SNP site T was used to cultivate taller kale plants.

5. The application according to claim 3, characterized in that, Kale with the SNP site C was used to cultivate short-height kale.

6. The application according to claim 3, characterized in that, The PCR amplification system consisted of: 2×Taq enzyme buffer, DNA template, CAPS-labeled primers shown in SEQ ID NO.6, CAPS-labeled primers shown in SEQ ID NO.7, and water; The volume ratio of 2×Taq enzyme buffer, DNA template, CAPS-labeled primers shown in SEQ ID NO.6, CAPS-labeled primers shown in SEQ ID NO.7, and water is 5:1:1:1:

2.

7. The application according to claim 3, characterized in that, The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 7 min; storage at 4°C.

8. The application according to claim 3, characterized in that, The enzyme digestion system is: PCR product, Hpy 166II restriction endonuclease, 10× digestion buffer, water; PCR products, Hpy The volume ratio of 166II restriction endonuclease, 10× digestion buffer, and water was 4:1:1:4.

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