InDel molecular marker of broccoli bolting time trait related gene BoMYB121 and application thereof

Abstract

This invention discloses genes related to the bolting time trait of broccoli. BoMYB121 This invention relates to the InDel molecular marker and its application, belonging to the field of molecular breeding technology. It is based on broccoli. BoMYB121 A 356 bp deletion variant located 721 bp downstream of the gene was used to develop an InDel molecular marker linked to the bolting time trait. PCR amplification using specific primer pairs resulted in a 635 bp specific band in materials carrying the deletion fragment and a 991 bp specific band in materials not carrying the deletion fragment. Validation on 244 broccoli germplasm accessions showed that 92.5% of the materials amplifying the 635 bp band were late bolting, while 70.1% of the materials amplifying the 991 bp band were early bolting. The molecular marker detection method provided by this invention is stable, easy to operate, and highly accurate, enabling rapid and precise identification of early / late bolting broccoli germplasm. It can be effectively applied to marker-assisted breeding of bolting-resistant broccoli varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to genes related to the bolting time trait of broccoli. BoMYB121 InDel molecular markers and their applications. Background Technology

[0002] Broccoli ( Brassica oleracea var. italica Broccoli is an important vegetable crop belonging to the Brassicaceae family and the Brassica genus. It is rich in nutrients and has high economic value. The timing of bolting is a key agronomical trait affecting the yield and quality of broccoli. Bolting too early can easily lead to poor head development, reduced yield, and lower quality. Therefore, bolting tolerance is one of the core objectives of broccoli breeding.

[0003] Bolting time is a quantitative trait controlled by multiple genes. The discovery of key genes regulating bolting time in broccoli and the development of functional markers are seriously insufficient. Traditional phenotypic identification is time-consuming, highly susceptible to environmental influences, and has low selection efficiency, which seriously restricts the breeding process of bolting-resistant broccoli varieties.

[0004] Developing specific molecular markers closely linked to the bolting time trait of broccoli enables precise early-generation screening and significantly improves breeding efficiency. Based on this, this invention identifies key regulatory genes for bolting time through genome-wide association analysis. BoMYB121 Furthermore, InDel molecular markers were developed to target downstream specific structural variations, providing an efficient tool for molecular breeding of broccoli to withstand bolting. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a trait related to the bolting time of broccoli. BoMYB121 This study investigates InDel gene molecular markers, specific primer pairs, identification methods, and their applications to address the problems of low efficiency and lack of efficient functional markers in the current identification of bolting time traits in broccoli.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an InDel molecular marker linked to the bolting time trait of broccoli, the molecular marker being located in broccoli BoMYB121 At a distance of 721 bp downstream of the gene, a 356 bp fragment is deleted. After amplification using specific primer pairs, materials carrying the deleted fragment produce a 635 bp specific band, of which late-bolting materials account for 92.5% (74 / 80); materials not carrying the deleted fragment produce a 991 bp specific band, of which early-bolting materials account for 70.1% (115 / 164).

[0007] The primer pairs used to amplify the above InDel molecular markers have the following nucleotide sequences: Forward primer SEQ ID No. 1; Reverse primer SEQ ID No.2.

[0008] This invention provides the application of the above-mentioned InDel molecular markers or primer pairs in the identification of bolting time traits of broccoli and in molecular marker-assisted breeding.

[0009] This invention provides a method for determining the timing of bolting in broccoli, comprising the following steps: (1) Extract genomic DNA from the broccoli sample to be tested; (2) PCR amplification of the genomic DNA using the primer pair described in claim 2; (3) Detect the fragment length of the PCR amplification product to determine the bolting type of broccoli.

[0010] Furthermore, in step (1), DNA extraction was performed using the CTAB method, with an OD260 / OD280 ratio of 1.8 to 2.0 and a concentration ≥20 ng / μL.

[0011] Furthermore, the 20 μL reaction system for PCR amplification in step (2) includes: 0.2 μM forward primer, 0.2 μM reverse primer, 50~100 ng DNA template, Taq enzyme and ddH2O; the PCR reaction program is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles; 72℃ final extension for 5 min.

[0012] Further, in step (3), the PCR products were detected by 1.5% agarose gel electrophoresis.

[0013] Furthermore, if a specific band of 635 bp is amplified, it is identified as a candidate late-bolting broccoli (accuracy of approximately 92.5%); if a specific band of 991 bp is amplified, it is identified as a candidate early-bolting broccoli (accuracy of approximately 70.1%).

[0014] A PCR kit for identifying bolting time traits in broccoli, the kit comprising the aforementioned specific primer pairs.

[0015] It contains at least the following beneficial technical effects: This invention is the first to reveal the properties of broccoli BoMYB121 The association between downstream fragment deletions of genes and bolting time traits was investigated, and a corresponding functional InDel marker was developed. This marker exhibits high stability and ease of operation, achieving a screening accuracy of 92.5% for late-bolting materials and 70.1% for early-bolting materials, both significantly higher than the random probability (50%) in natural populations. It can be effectively used for large-scale screening of early / late bolting germplasm in broccoli, significantly improving the efficiency of molecular marker-assisted breeding. Attached Figure Description

[0016] Figure 1 Phenotypic diagram of early-bolting and late-bolting broccoli materials sown and transplanted at the same time.

[0017] Figure 2 Agarose gel electrophoresis images of the molecular marker PCR amplification products of this invention (M: Marker; 991bp for early bolting material; 635bp for late bolting material).

[0018] Figure 3 Sanger sequencing comparison of PCR products from early-bolting and late-bolting materials. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0025] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0026] Example 1: Genes regulating bolting time in broccoli BoMYB121 Discovery and Development of InDel Molecular Markers Test materials The test materials consisted of 244 broccoli inbred lines provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences; they were grown at the farm of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (No. 12, Zhongguancun South Street, Haidian District, Beijing) and were managed under routine conditions.

[0027] Bolting phenotypic identification criteria: Bolting was defined as the diameter of the bud reaching 1 cm. The number of days from transplanting to bolting was recorded to classify plants into early bolting (≤34 days from transplanting) and late bolting (≥42 days from transplanting). The phenotypic differences between early and late bolting materials sown and transplanted at the same time are shown in Figure 1.

[0028] Genome-wide association study (GWAS) GWAS analysis was performed using an MLM model, with a significance threshold. lg(P)≥5; on chromosome 9 of broccoli BoMYB121 A site with a highly significant association with bolting time was detected in the gene region.

[0029] Gene structural variation detection PCR amplification and Sanger sequencing were performed on early and late bolting materials. The results showed that 92.5% of the materials with a 356 bp deletion were late bolting materials, while 70.1% of the materials without this deletion were early bolting materials.

[0030] Specific primer design Design primer pairs for the missing regions: Forward primer (F): 5'-TCGCAAAAGTTAGACAAGTTAAAGTC-3' (SEQ ID No. 1); Reverse primer (R): 5'-CAAATCAAAACCCAAACTAAATGAGT-3' (SEQ ID No. 2); Example 2: Molecular Identification Method for Early / Late Bolting of Broccoli DNA extraction and detection Genomic DNA was extracted from broccoli using the CTAB method; integrity was assessed by agarose gel electrophoresis; and the concentration was determined by micro-spectrophotometer: OD260 / 280 = 1.8~2.0, concentration ≥20 ng / μL.

[0031] PCR amplification system and procedure 20μL system: 0.2μM forward primer, 0.2μM reverse primer, 50~100ng template DNA, 2×TaqPCRStarMix, ddH2O to make up.

[0032] Reaction program: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 32 cycles; 72℃ extension for 5 min.

[0033] Electrophoretic detection Electrophoresis was performed on a 1.5% agarose gel at 180V for 20 min using 1×TAE buffer; the bands were observed using a gel imaging system.

[0034] Classification criteria A 635bp band was amplified: identified as a candidate late-bolting broccoli (accuracy 92.5%).

[0035] A 991bp band was amplified: it was identified as a candidate early bolting broccoli (accuracy 70.1%).

[0036] Example 3 Molecular marker verification Validation was performed on 244 broccoli germplasm resources. Results showed that 80 accessions amplified a 635 bp specific band (i.e., a 356 bp deletion), of which 74 were late-bolting accessions and 6 were early-bolting accessions, with late-bolting accessions accounting for 92.5%. The remaining 164 accessions amplified a 991 bp band (without deletion), of which 115 were early-bolting accessions, accounting for 70.1%. These results indicate that this molecular marker can be effectively used for two-way screening of early-bolting and late-bolting broccoli germplasm.

[0037] Sanger sequencing results ( Figure 3 This confirmed that all materials that amplified a 635 bp band had a 356 bp missing fragment, which was completely consistent with the electrophoresis typing results.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An InDel molecular marker associated with bolting time trait of broccoli, characterized in that, The molecular marker is located in broccoli. BoMYB121 The gene is produced by a 356bp deletion at 721bp downstream. After amplification using specific primers, materials carrying the deleted fragment produced a 635 bp specific band, of which late-bolting materials accounted for 92.5%; materials not carrying the deleted fragment produced a 991 bp specific band, of which early-bolting materials accounted for 70.1%.

2. A primer pair for amplifying the InDel molecular marker of claim 1, characterized in that, The nucleotide sequences of the primer pair are as follows: Forward primer SEQ ID No. 1; Reverse primer SEQ ID No.

2.

3. The application of the InDel molecular marker of claim 1 or the primer pair of claim 2 in the identification of bolting time trait of broccoli and molecular marker-assisted breeding.

4. A method for determining the timing of bolting in broccoli, characterized in that, Includes the following steps: (1) Extract genomic DNA from the broccoli sample to be tested; (2) PCR amplification of the genomic DNA using the primer pair described in claim 2; (3) Detect the fragment length of the PCR amplification product to determine the timing of bolting of broccoli.

5. The method according to claim 4, characterized in that, In step (1), DNA extraction was performed using the CTAB method, with an OD260 / OD280 ratio of 1.8 to 2.0 and a concentration ≥20 ng / μL.

6. The method according to claim 4, characterized in that, The 20 μL reaction system for PCR amplification in step (2) includes: 0.2 μM forward primer, 0.2 μM reverse primer, 50~100 ng DNA template, Taq enzyme and ddH2O; the PCR reaction program is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles; 72℃ final extension for 5 min.

7. The method according to claim 3, characterized in that, Step (3) uses 1.5% agarose gel electrophoresis to detect PCR products.

8. The method according to claim 4 or 7, characterized in that, If a specific band of 635 bp is amplified, it is identified as a candidate late-bolting broccoli; if a specific band of 991 bp is amplified, it is identified as a candidate early-bolting broccoli.

9. A PCR kit for identifying bolting time characteristics of broccoli, characterized in that, The kit contains the specific primer pair as described in claim 2.

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