Molecular markers associated with brussel sprouts tolerance to waterlogging and applications

Abstract

The application belongs to the technical field of crop molecular marker, and particularly discloses a molecular marker related to the tolerance of Brassica napus to waterlogging and application. Bna.C08.GUN4 The application identifies a 1.2 Kb and a 2.6 Kb transposon insertion located in the gene promoter region by GWAS, and the Brassica napus containing the two transposon insertions has high waterlogging tolerance. The molecular marker of the application is used for the assisted selection of Brassica napus with high waterlogging tolerance, and whether the Brassica napus germplasm is waterlogging-tolerant can be predicted by detecting the characteristic bands of the molecular marker. The identification method is simple and feasible, the selection efficiency is high, and the application has great application potential in the field of waterlogging tolerance improvement and breeding of Brassica napus.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop molecular markers, and in particular relates to a molecular marker related to waterlogging tolerance of Brassica napus and its application. Background Art

[0002] Rapeseed is one of the world's most important oilseed crops, accounting for approximately 50% of my country's vegetable oil supply. Waterlogging stress reduces the activity of photosynthesis-related enzymes, hinders chlorophyll synthesis and causes degradation, inhibits the formation of new leaves, and causes leaves to wilt, yellow, senesce, and fall off, ultimately impacting plant growth and development, and even leading to plant death. Waterlogging also leads to losses in seed yield during the mature stage of rapeseed production, severely impacting its production.

[0003] At present, the research on rapeseed's response to waterlogging mainly focuses on growth and physiological responses during stress, growth inhibition during maturity, etc. In recent years, comparative transcriptome and proteome analysis has been the main means to analyze the genetic basis of rapeseed's response to waterlogging. With the widespread application of cutting-edge technologies such as genomics and phenotypic methods in crop research, the study of rapeseed's response to waterlogging has also ushered in new opportunities. Genome-wide association analysis (GWAS) refers to a method of using molecular markers near genes or genes to perform association analysis with one or more complex traits at the whole genome level to find genetic loci associated with the target traits. It has now become a key means of identifying important agronomic traits.

[0004] Most of the previous studies focused on the physiological and morphological characteristics of rapeseed's response to waterlogging. Although the current research on plant waterlogging tolerance has progressed rapidly, most of them have stagnated at the stage of initial QTL positioning and excavation, while the research content of identifying functional genes and exploring their molecular mechanisms is relatively limited, and no waterlogging tolerance sites or key genes have been cloned so far. Therefore, in-depth research on rapeseed's response to waterlogging and its regulatory mechanisms, the discovery of genetic sites and genes that regulate waterlogging responses, and the identification of germplasm with excellent waterlogging tolerance will help deepen the understanding of rapeseed's waterlogging response mechanism and provide a theoretical basis and germplasm resources for genetic improvement for the cultivation of new waterlogging-tolerant rapeseed varieties. The present invention uses GWAS based on structural variation (SV) to identify two adjacent transposon insertions of 1.2Kb and 2.6Kb in waterlogging-tolerant rapeseed germplasm, located in the promoter region of the Bna.C08.GUN4 gene. By designing primers targeting the TE insertion, molecular markers closely linked to waterlogging tolerance are developed for molecular-assisted selection of offspring with strong waterlogging tolerance in rapeseed breeding. Summary of the Invention

[0005] The present invention measured the SPAD values ​​of 350 rapeseed germplasms planted at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences in Wuhan in 2023 after waterlogging treatment, and combined with resequencing data to conduct GWAS analysis based on structural variation. In the waterlogging-tolerant rapeseed, a 1.2Kb and a 2.6Kb transposon insertion were identified in the promoter region of the Bna.C08.GUN4 gene on chromosome C08. Molecular markers related to waterlogging tolerance were developed for waterlogging tolerance identification and rapeseed breeding.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A molecular marker for identifying waterlogging tolerance in Brassica napus, comprising two 1.2 Kb and 2.6 Kb transposon insertions located in the promoter region of the Bna.C08.GUN4 gene, with sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] A kit for identifying waterlogging tolerance in Brassica napus contains reagents for detecting molecular markers of sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2. In a specific embodiment of the present invention, primers of sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used to simultaneously detect the molecular markers.

[0009] A method for identifying waterlogging tolerance in Brassica napus includes detecting whether the rapeseed contains molecular markers represented by sequences set forth in SEQ ID NOs. 1 and 2. Rapeseed plants containing these molecular markers exhibit high waterlogging tolerance. In a specific embodiment of the present invention, the markers are amplified using primers represented by sequences set forth in SEQ ID NOs. 3 and 4. If a 4.4 kb product is amplified, the plant exhibits high waterlogging tolerance, while if a 537 bp product is amplified, the plant exhibits low waterlogging tolerance.

[0010] Compared with existing technologies, the present invention can identify rapeseed varieties with strong waterlogging tolerance by using conventional PCR and agarose gel analysis to detect the presence of the target insert. This identification method is simple, feasible, and highly efficient. It can identify rapeseed varieties with strong waterlogging tolerance in the early stages of their growth, providing a clear selection target and unaffected by environmental factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 (a) The frequency distribution diagram of SPAD values ​​of 350 materials in the control group and (b) the frequency distribution diagram of SPAD values ​​of 350 materials in the waterlogging group.

[0012] Figure 2Results of SV-GWAS data analysis. a shows the relative SPAD values ​​of 350 accessions in the genome-wide association analysis, and b shows the locations of the two identified transposons on chromosome C08.

[0013] Figure 3 The relative SPAD value significance analysis of 350 associated groups. INS is a genotype containing 1.2 Kb and 2.6 Kb transposon insertions, and GUN4 is a genotype without 1.2 Kb and 2.6 Kb transposon insertions.

[0014] Figure 4 The genotype identification results of 5 waterlogging-tolerant and 5 waterlogging-intolerant rapeseed varieties (the marker used was 5K).

[0015] Figure 5 The growth of the control group and the waterlogging group after 7 days of waterlogging treatment.

[0016] Figure 6 Two genotypes (GUN4 and GUN4) before and after waterlogging treatment INS ) Relative expression level of BnaC08.GUN4 gene in materials.

[0017] Figure 7 The two genotypes (GUN4 and GUN4) were treated with waterlogging for 7 days. INS )Measurement results of physiological and biochemical indicators of materials.

[0018] Figure 8 There are two genotypes (GUN4 and GUN4 INS ) materials’ promoter activities were compared. DETAILED DESCRIPTION

[0019] The present invention is further illustrated below with reference to the following examples. Unless otherwise specified, the experimental methods in the following examples are conventional experimental methods. The experimental reagents and consumables described in the following examples can be purchased from commercial companies unless otherwise specified. In this example, the detailed steps for screening for molecular markers in rapeseed materials for waterlogging tolerance are as follows:

[0020] 1. Rapeseed related population planting and SPAD value determination

[0021] 350 germplasm resource groups collected by the inventor's research group in the early stage were used as experimental materials. They were planted at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences in Wuhan in 2023 and divided into a control group and a waterlogged experimental group. Each group was repeated three times, and each material was planted in double rows with a row length of 1.5m and a row spacing of 0.25m. The waterlogged treatment group began to be waterlogged when the rapeseed reached the five-leaf stage one month after sowing. Field irrigation was used, and the water level needed to be about 1-2cm above the soil surface, and waterlogging continued for ten days; the control group maintained conventional field management conditions during the growth period. After the waterlogged group was waterlogged for ten days, six plants with the same growth were selected from each material in the control group and the experimental group, and the SPAD value was measured using a SPAD instrument (SPAD-502Plus). The average value was calculated to obtain the final SPAD value of each material.

[0022] 2. Data Analysis

[0023] SV-GWAS analysis was performed using relative SPAD values ​​(SPAD value of the waterlogging experimental group / SPAD value of the control group). Combining 350 resequencing data, a 1.2 Kb (SEQ ID NO. 1) and a 2.6 Kb (SEQ ID NO. 2) transposon insertion were identified on chromosome C08, located in the promoter region of the Bna.C08.GUN4 gene ( Figure 2 ), and Bna.C08.GUN4 is known to promote chlorophyll synthesis by activating magnesium chelatase activity. A 700-bp sequence insertion was also identified on chromosome A10, located in the promoter region of the Bna.A10.DRM2 gene. However, the Bna.A10.DRM2 gene has not yet been functionally annotated, and SV-GWAS analysis results did not show a clustered increase compared to the C08 chromosome locus, so further analysis has not yet been performed.

[0024] 3.TE fragment amplification

[0025] The CTAB method was used to extract 350 genomic DNA samples from the associated population, and the DNA quality was tested by 1% agarose gel electrophoresis. Primers were designed to amplify the two transposons inserted into the C08 chromosome. The primer sequences are shown in SEQ ID NO. 3 and 4. The PCR reaction system is shown in Table 1, and the PCR amplification reaction procedure is shown in Table 2. The amplified products were detected by 1% agarose gel electrophoresis. After all the tests were completed, the two genotype materials were distinguished and the relative SPAD value significance was calculated. Figure 3 As shown, GUN4 contains two transposon insertions INS The relative SPAD value of the genotype was significantly higher than that of the GUN4 genotype without transposon insertion.

[0026] Table 1 PCR reaction system

[0027] Components Volume (μL) 2x Phanta Flash Master Mix(Dye Plus) 10 Forward primer 1 Reverse primer 1 <![CDATA[ddH2O]]> 7 DNA 1

[0028] Table 2 PCR amplification reaction program

[0029]

[0030] 4. Identification of two genotypes and phenotypes and determination of physiological and biochemical indicators

[0031] Based on the data analysis results and the relative SPAD values, 5 materials containing two transposon insertions (8S006, 8S007, 8S091, 8S149, 8SA71) and 5 materials without transposon insertions (8S053, 8S190, 8S245, 8S292, 8SA59) were selected for the determination of physiological and biochemical indicators. The genotype identification results of the 10 materials are as follows: Figure 4 shown.

[0032] 4.1 Evaluation of indoor stain resistance of representative materials

[0033] Ten portions of material seeds were divided into 2mL centrifuge tubes and planted in 8L nutrient pots, with 40 seeds planted for each portion of material. The solution used was Hoagland's nutrient solution. Cultured in the dark on the first day of sowing, the cover was uncovered after the radicle was exposed, and the light was turned on to maintain normal growth, with light: dark = 16:8h. After growing for a week, transplanted into 5×10 soil culture trays, with ten seedlings transplanted for each portion of material. The soil used was nutrient soil purchased from the laboratory. After a week of normal growth, water was started when the seedlings grew to the two-leaf to three-leaf stage. The treatments were divided into a control group and a waterlogged group. The control group maintained normal growth conditions during the growth process, while the waterlogged group maintained the water level 1-2cm above the soil surface during the growth process. Waterlogging was continued for 7 days. After 7 days, the growth of the control group and the waterlogged group was as follows. Figure 5 As shown. Figure 5 It can be seen that there is no difference in the growth of the control group of the two genotype materials, but after 7 days of waterlogging, GUN4 INS Compared with the GUN4 genotype, the plant growth and leaf greenness of this genotype are stronger.

[0034] The young leaves of the control group and the treatment group of each material were selected to extract plant tissue RNA. After RNA reverse transcription, qRT-PCR experiments were performed. The relative expression levels of the BnaC08.GUN4 gene before and after waterlogging were detected by comparing the waterlogged group with the control group. Figure 6 As shown, GUN4 INS After waterlogging, the expression of BnaC08.GUN4 gene in the GUN4 genotype material showed an upward trend compared with the control group, while the expression of BnaC08.GUN4 gene in the GUN4 genotype material showed a downward trend; INSAmong the materials with the same genotype, the relative expression level of BnaC08.GUN4 gene was significantly higher than that of GUN4 genotype.

[0035] Three plants with consistent growth were selected and the SPAD values ​​were measured using a SPAD instrument (SPAD-502Plus). Three plants with consistent growth were selected and the aboveground fresh weight was weighed. Three tissue samples of 0.2 g were weighed for malondialdehyde determination, and three tissue samples of 0.2 g were weighed for chlorophyll content determination.

[0036] like Figure 7 As shown in the figure, under the control condition (without waterlogging treatment), there was no significant difference in chlorophyll content, SPAD value, aboveground fresh weight and MDA content between the two genotypes. However, under waterlogging treatment, GUN4 containing transposon insertion had a significant difference in chlorophyll content, SPAD value, aboveground fresh weight and MDA content. INS The chlorophyll content, SPAD value and aboveground fresh weight of the genotype material were significantly higher than those of the GUN4 genotype material, and the MDA content was significantly lower than that of the GUN4 genotype material; this shows that the molecular markers developed by the present invention can be used to identify whether the rapeseed variety is a material with good waterlogging tolerance.

[0037] 4.2 Promoter activity of representative materials

[0038] In order to verify whether the insertion of TE has an effect on promoter activity, we amplified 2Kb fragments of the promoter region of the two genotypes and connected them to the pGreenII-0800-LUC vector. After transforming Escherichia coli DH5α, we performed single clone identification, extracted the plasmids of Escherichia coli from the positive clones and transferred them into Agrobacterium GV3101. Then, Agrobacterium tumefaciens strains (GV3101) carrying different plasmids were injected into Nicotiana benthamiana leaves. Within 48-72h, luciferase reaction solution was evenly applied on the back of the tobacco, reacted for about 5 minutes under dark conditions, and LUC fluorescence was observed under a live imaging device. At least six independent leaves were used for each experiment. Figure 8 As shown, GUN4 contains two transposon insertions INS The ability of the promoter of the genotype material to drive reporter gene expression is higher than that of the promoter of the GUN4 genotype material.

Claims

1. A molecular marker for identifying waterlogging tolerance in Brassica napus, characterized in that: The sequences of the molecular markers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A kit for identifying waterlogging tolerance of Brassica napus, characterized in that: The kit contains reagents for detecting the molecular marker according to claim 1.

3. The kit according to claim 2, wherein The primer sequences for detecting the molecular marker according to claim 1 are shown in SEQ ID NOs. 3 and 4.

4. Use of the molecular marker according to claim 1 or the kit according to claim 2 or 3 in identifying waterlogging tolerance of rapeseed or breeding waterlogging-tolerant rapeseed.

5. The use according to claim 4, characterized in that The rapeseed containing the molecular marker according to claim 1 has strong waterlogging tolerance.

6. A method for identifying waterlogging tolerance of Brassica napus, characterized in that: The method comprises detecting whether the rapeseed to be tested contains the molecular marker according to claim 1, and the rapeseed containing the molecular marker according to claim 1 has high waterlogging tolerance.

7. The method according to claim 6, characterized in that The molecular marker according to claim 1 is amplified using primers, wherein the primer sequences are shown in SEQ ID NOs. 3 and 4. If a 4.4 Kb product is obtained by amplification, it has high waterlogging resistance; if a 537 bp product is obtained by amplification, it has low waterlogging resistance.

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