InDel molecular marker related to fragrance of non-heading Chinese cabbage and application of InDel molecular marker
By developing InDel molecular markers and their detection primers related to the aroma of non-heading Chinese cabbage, and utilizing PCR and gel electrophoresis techniques, the problem of improving the aroma quality of non-heading Chinese cabbage was solved, enabling rapid and accurate material screening and improving breeding efficiency.
Patent Information
- Application Number
- CN202511407608.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2026-01-02
AI Technical Summary
In the current technology, there has been little progress in improving the aroma quality of non-heading Chinese cabbage. There is a lack of effective molecular marker-assisted breeding methods, making it difficult to quickly distinguish between aromatic and non-aromatic materials.
An InDel molecular marker closely linked to the aroma trait of non-heading Chinese cabbage was developed, along with its detection primers. The aroma type of the plant was detected by PCR amplification and polyacrylamide gel electrophoresis. The aroma type, non-aroma type, and heterozygous type were distinguished by the band length of the PCR amplification product using specific primers.
This technology enables rapid and accurate differentiation between aromatic and non-aromatic non-heading Chinese cabbage varieties, improving breeding efficiency and shortening the breeding cycle.
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Figure CN121249941A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of molecular marker assisted breeding, and particularly relates to an InDel molecular marker related to aroma trait of Brassica campestris ssp. chinensis, detection primers and application. BACKGROUND
[0002] Aroma plays a vital role in the sensory appeal and enjoyment of food, and significantly influences the value and consumption of agricultural products. For example, aromatic rice varieties are known for their unique aroma, and their price is almost twice that of ordinary rice on the market. Therefore, improving the aromatic trait has become an important goal of breeding programs.
[0003] Brassica campestris ssp. chinensis, as a cruciferous vegetable with green leaves as the product, is one of the most popular vegetables in southern China, and is also the main leafy vegetable in large and medium-sized cities in the middle and lower reaches of the Yangtze River. The aroma of Brassica campestris ssp. chinensis is an important indicator for evaluating its quality. Since Brassica campestris ssp. chinensis is difficult to directly smell the rich fruit aroma like melon and strawberry, and also does not release obvious aroma after being cut or broken like cucumber, the research on flavor substances of Brassica campestris ssp. chinensis is less; in addition, there are few germplasm resources with obvious aroma, and the improvement of Brassica campestris ssp. chinensis aroma quality has made little progress. Therefore, the development of aroma molecular marker assisted breeding will provide important support for improving the aroma quality of Brassica campestris ssp. chinensis and breeding new varieties of Brassica campestris ssp. chinensis. SUMMARY
[0004] The purpose of the present application is to provide a molecular marker and detection primers closely linked to the aroma trait of Brassica campestris ssp. chinensis and application.
[0005] In order to achieve the purpose of the present application, the technical scheme of the present application is as follows:
[0006] An InDel molecular marker related to the aroma of Brassica campestris ssp. chinensis, the nucleotide sequence of which is AGAGAC, the InDel molecular marker is located at the Chr7: 28389774-28389951 site of the whole genome of Brassica campestris ssp. chinensis, and the reference genome version is Pakchoi-XQC-v1.0 (http: / / www.tbgr.org.cn). When the InDel molecular marker sequence is contained, the Brassica campestris ssp. chinensis has aroma, and when the InDel molecular marker sequence is deleted or is a heterozygous type, the Brassica campestris ssp. chinensis does not have aroma.
[0007] Table 1 Site information of InDel11 marker
[0008] marker name chromosome amplified fragment region insertion / deletion fragment size InDel11 Chr7 28389774-28389951 6bp
[0009] The marker position in Table 1 is determined according to the whole genome sequence of Brassica rapa (Liu) et al. (Pakchoi-XQC-v1.0, http: / / www.tbgr.org.cn)
[0010] Further, the present application provides a detection primer for detecting the molecular marker.
[0011] The detection primer comprises a forward primer F and a reverse primer R, and the specific information is as follows:
[0012] The forward primer Chr7-28389774-indel-F: 5'-TCAAAGTCGGTAGTATTTGC-3' (SEQ ID NO. 1),
[0013] The reverse primer Chr7-28389774-indel-R: 5'-ATCTAAGGAGAGCTGTACTG-3' (SEQ ID NO. 2).
[0014] A kit containing the specific primer pair.
[0015] The specific primer pair or the kit is applied in distinguishing the aroma or non-aroma material of the seedling stage of Brassica rapa.
[0016] The application comprises the following steps:
[0017] (1) extracting the DNA of the Brassica rapa sample to be detected;
[0018] (2) using the genomic DNA extracted in step (1) as a template, using ordinary Taq enzyme and the aforementioned detection primer pair to perform PCR amplification on the molecular marker;
[0019] (3) detecting the PCR amplification product by polyacrylamide gel electrophoresis.
[0020] Further, the method of the application is as follows: using the genomic DNA of the Brassica rapa to be detected as a template, using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 to perform PCR amplification, using polyacrylamide gel to detect the PCR product, if a 178 bp band is amplified, the plant is of the aroma type; if a 172 bp band is amplified, the plant is of the non-aroma type; if two bands of 178 bp and 172 bp are amplified, the plant is of the non-aroma type.
[0021] The specific primer pair or the kit of claim 3 is applied in marker-assisted breeding of Brassica rapa with aroma.
[0022] Further, the genomic DNA of the Brassica rapa L. to be detected is used as a template, and a primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 is used for PCR amplification, and the PCR product is detected by using a polyacrylamide gel, if a band of 178 bp is amplified, it is indicated that the plant is a homozygous type containing the InDel molecular marker, and is an aromatic material; if a band of 172 bp is amplified, it is indicated that the plant is a homozygous type lacking the InDel molecular marker, and is a non-aromatic material; if two bands of 178 bp and 172 bp are amplified, the plant is a heterozygous type, and is a non-aromatic material.
[0023] A molecular marker method for screening aromatic Brassica rapa L. materials at the seedling stage, comprising extracting the genomic DNA of the Brassica rapa L. to be detected, using the genomic DNA as a template, and using a primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 to perform PCR amplification on the molecular marker; the PCR amplification product is detected by polyacrylamide gel electrophoresis, if a band of 178 bp is amplified, the plant is aromatic; if a band of 172 bp is amplified, the plant is non-aromatic; if two bands of 178 bp and 172 bp are amplified, the plant is non-aromatic.
[0024] In the detection method, the PCR reaction system is as follows: in a 10 μL reaction system, 3 μL deionized water, 0.5 μL of 10 μM Chr7-28389774-indel-F / R primer each, 1 μL of 1 template DNA, and 5 μL of 2×Magic Green TaqSuperMix.
[0025] Further, in step (2), the PCR reaction conditions are as follows: 95℃, 3min; 95℃, 15s; 55℃, 15s; 72℃, 10s; 35 cycles; 72, 5min; 4℃ storage.
[0026] Further, in step (3), the configuration system of the polyacrylamide gel electrophoresis is as follows: in a 61 mL gel system, 10×TBE 6 mL, 30% PA 15 mL, TEMED 90 μL, 10% APS 400 μL, and deionized water 40 mL.
[0027] Further, in step (3), the silver staining and developing system of the polyacrylamide gel is as follows: 2 g AgNO3 / 1000 mL H2O staining solution, 3 g NaOH / 200 mL H2O-2 mL formaldehyde stock solution developing solution.
[0028] Further, in step (3), the silver staining and developing reaction procedure of the polyacrylamide gel is as follows: nitrate solution is used for light staining for 10 minutes, and then distilled water is used for rinsing for 3 times, each time for 1 minute. After rinsing, the polyacrylamide gel is placed in a sodium hydroxide-formaldehyde developing solution, and then is slightly shaken on a horizontal shaker for 5 minutes.
[0029] Beneficial effects:
[0030] The application is based on sequence-based insertion / deletion fragment-related primers for identifying aroma traits of Brassica chinensis. The technology can detect multiple samples in a high-throughput manner, can quickly distinguish between aroma-containing and aroma-free materials of Brassica chinensis, and is simple to operate and highly accurate. The molecular marker provided by the application can greatly accelerate the breeding of aromatic varieties of Brassica chinensis, shorten the breeding period, and improve the breeding efficiency.
[0031] The raw materials or reagents used in the application are all ordinary commercially available products, and the operations involved are all conventional operations in the field unless otherwise specified.
[0032] On the basis of common sense in the art, the above-mentioned preferred conditions can be combined with each other to obtain a specific embodiment. BRIEF DESCRIPTION OF DRAWINGS
[0033] Figure 1 Results of rapid positioning of aroma genes of Brassica chinensis by using the BSA method.
[0034] Figure 2 Polyacrylamide gel electrophoresis result diagram of InDel11 molecular marker after amplification in Brassica chinensis aroma-free variety Suzhouqing, Brassica chinensis aroma-containing variety Xiangqincan, F1 material obtained by crossing the two materials, and F2 material obtained by selfing the F1 material. Among them, M is DL500 Marker, 1-45 is aroma-containing F2 material, and 46-87 is aroma-free F2 material. DETAILED DESCRIPTION
[0035] The preferred embodiments of the application will be described in detail below with reference to the examples. It should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the application. Those skilled in the art can make various modifications and replacements to the application without departing from the spirit and principles of the application.
[0036] In the following examples, the experimental methods used are conventional methods unless otherwise specified.
[0037] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.
[0038] The primers used in the application are synthesized by Beijing Qikexin Biotechnology Co., Ltd.
[0039] Example 1 Determination of candidate regions and development of linked markers associated with aroma trait in Brassica rapa L. using BSA method
[0040] 1. Construction of Brassica rapa genetic population
[0041] The F1 generation was obtained by crossing the high-generation inbred line with aroma Brassica rapa ‘Xiangqincan’ as the female parent and the non-aroma Brassica rapa ‘Suzhouqing’ as the male parent. The F2 separation population was obtained by selfing the F1 generation.
[0042] 2. Re-sequencing of F2 plants with aroma and non-aroma
[0043] The leaves of 30 plants with aroma and 30 plants without aroma were taken (preliminary identification of whether the materials had aroma was made by smelling in the field by 15 team members, and further determination of the samples to be tested was made by GC-MS detection), and DNA was extracted using the CTAB method. The extracted DNA samples were sequenced by China Hangzhou Biotechnology Co., Ltd. using the Illumina NovaSeq 6000 platform to produce 150 bp double-end raw data.
[0044] 3. BSA-seq data analysis and association analysis
[0045] To ensure accurate and reliable analysis results, the raw data was obtained after removing low-quality sequences and sequencing adapter sequences. The filtered double-end sequencing data was aligned to the Brassica rapa genome of version Pakchoi-XQC-v1.0 using the BWA alignment software (Burrows–Wheeler Aligner, v0.7.13). After alignment, GATK HaplotypeCaller (version 4.0.4.0) was used to detect SNPs and InDels for each sample. Next, the SNP-index of the offspring pool and the difference were calculated, and regions with extremely significant differences in SNP-index between the two offspring pools were selected, and the target trait region was located on the chromosome (i.e., to determine which segment on the chromosome may be associated with the aroma trait).
[0046] 4. Preliminary mapping of aroma trait genes
[0047] Based on the results of association analysis and evaluation of the regions associated with the aroma trait of Brassica rapa, the interval was located to the Chr7:27730000-30930000 interval 3.20 Mb according to the ΔSNP-index and ED two algorithms. Figure 1 )
[0048] 5. Fine mapping of aroma trait and development of linked markers
[0049] In order to narrow the initial region size, combined with the data of Brassica rapa genome, eight polymorphic InDel markers were developed in the initial region, and these markers were used to screen recombinant single plants in F2 population to narrow the candidate region, and finally the aroma trait gene was located between 28152596-28402633. Further screening of F 2：3 Single plant narrowing interval, four polymorphic InDel markers were designed in the candidate region, among which InDel 11 was linked to Brassica rapa aroma trait.
[0050] 6. Synthesis of InDel 11 marker primer
[0051] According to the characteristics of InDel variation information, specific PCR primers were designed, including forward primers and reverse primers.
[0052] The primer sequences are as follows:
[0053] Forward primer F: 5'-TCAAAGTCGGTAGTATTTGC-3'(SEQ ID No. 1),
[0054] Reverse primer R: 5'-ATCTAAGGAGAGCTGTACTG-3'(SEQ ID No. 2).
[0055] The above primer sequences were synthesized by Beijing Qikexing Biotechnology Co., Ltd.
[0056] Example 2 Indel 11 molecular marker detection of F2 population and two parents, F1
[0057] The detection method is as follows:
[0058] 1) Extraction of plant genomic DNA
[0059] Take the leaves of the Brassica rapa to be tested, and extract the whole genome DNA by CTAB method. In this F2 population, 45 plants with aroma and 42 plants without aroma (independent of the F2 population samples in Example 1) were selected for detection.
[0060] 2) PCR amplification of InDel 11 marker using ordinary Taq enzyme, and the amplification system is as follows:
[0061]
[0062] 3) The PCR reaction conditions are as follows:
[0063] 95℃, 3min; 95℃, 15s; 55℃, 15s; 72℃, 10s; 35 cycles; 72, 5min; 4℃ storage.
[0064] 4) PCR products were detected by polyacrylamide gel
[0065] The amplified products were detected by polyacrylamide gel electrophoresis. If a band of 178 bp was amplified, the plant was of the aromatic type; if a band of 172 bp was amplified, the plant was of the non-aromatic type; if bands of 178 bp and 172 bp were amplified, the plant was of the non-aromatic type. Figure 2
[0066] Results analysis: The InDel11 marker genotyping results were completely consistent with the field investigation results. The results showed that the InDel11 marker could accurately distinguish the aromatic and non-aromatic types of Brassica chinensis, and was fast, accurate, and could be used for marker-assisted selection breeding.
[0067] Although the present application has been described in detail by the general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection required by the present application.
Claims
1. An InDel molecular marker associated with the aroma of non-heading Chinese cabbage, characterized in that, The nucleotide sequence is AGAGAC. The InDel molecular marker is located at Chr7:28389774-28389951 in the whole genome of non-heading Chinese cabbage, with the reference genome version being Pakchoi-XQC-v1.
0. When this InDel molecular marker sequence is present, non-heading Chinese cabbage has an aroma; when this InDel molecular marker sequence is missing or is heterozygous, non-heading Chinese cabbage does not have an aroma.
2. A specific primer pair for identifying the InDel molecular marker as described in claim 1, characterized in that, The forward primer is shown in SEQ ID NO.1, and the reverse primer is shown in SEQ ID NO.
2.
3. A kit containing the specific primer pair as described in claim 2.
4. The application of the specific primer pair of claim 2 or the kit of claim 3 in distinguishing between aromatic and non-aromatic Chinese cabbage seedlings that do not form heads.
5. The application according to claim 4, characterized in that, The application method is as follows: using the genomic DNA of the non-heading Chinese cabbage to be tested as a template, PCR amplification is performed using the primer pair shown in SEQ ID No. 1 and SEQ ID No.
2. The PCR product is detected using polyacrylamide gel electrophoresis. If a 178bp band is amplified, the plant is an aromatic type; if a 172bp band is amplified, the plant is an unaromatic type; if two bands of 178bp and 172bp are amplified, the plant is an unaromatic type.
6. The application of the specific primer pair of claim 2 or the kit of claim 3 in molecular marker-assisted breeding for selecting aromatic non-heading Chinese cabbage.
7. The application according to claim 6, characterized in that, Using the genomic DNA of the non-heading Chinese cabbage to be tested as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 1 and SEQ ID No.
2. The PCR products were detected using polyacrylamide gel electrophoresis. If a 178bp band was amplified, it indicated that the plant was homozygous with the InDel molecular marker described in claim 1 and was an aromatic material; if a 172bp band was amplified, the plant was homozygous without the InDel molecular marker described in claim 1 and was an aromaless material; if two bands of 178bp and 172bp were amplified, the plant was heterozygous and was an aromaless material.
8. A molecular marker method for screening aromatic, non-heading Chinese cabbage materials during the seedling stage, characterized in that, The procedure includes extracting genomic DNA from the non-heading Chinese cabbage to be tested, using this DNA as a template to perform PCR amplification of the molecular marker described in claim 1 using the primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2; detecting the PCR amplification product by polyacrylamide gel electrophoresis, if a 178bp band is amplified, the plant is an aromatic type; if a 172bp band is amplified, the plant is an unaromatic type; if two bands of 178bp and 172bp are amplified, the plant is an unaromatic type.
9. The method according to claim 8, characterized in that, The PCR reaction system is as follows: in a 10 μL reaction system, there is 3 μL of deionized water, 0.5 μL each of 10 μM Chr7-28389774-indel-F / R primers, 1 μL of template DNA, and 5 μL of 2×Magic Green Taq SuperMix.
10. The method according to claim 8, characterized in that, The PCR reaction conditions were as follows: 95℃ for 3 min; 95℃ for 15 s; 55℃ for 15 s; 72℃ for 10 s; 35 cycles; 72℃ for 5 min; and stored at 4℃.