An SSR molecular marker related to the content of 3-butenyl glucosinolate in Brassica rapa crops and its application

Abstract

The present invention discloses an SSR molecular marker related to the content of 3-butenyl glucosinolate in Brassica crops and its application, belonging to the field of molecular markers. In the present invention, an F2-derived population constructed with 'A01-5' and '59-1' as parents is used, and QTL linkage analysis is carried out by combining the genotypes and target phenotypic traits of individual plants in the F2-derived population. As a result, two new molecular markers, GLS47-8 and GLS34-12, which are tightly linked to the target traits, are found. After verification, the molecular markers GLS47-8 and GLS34-12 can accurately identify the content of 3-butenyl glucosinolate in Brassica crops, providing new molecular markers for the screening and breeding of Brassica crops with low glucosinolate content, and having important guiding significance for improving the glucosinolate components of Brassica crops and applying them to low-glucosinolate breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular markers, and particularly to an SSR molecular marker related to the content of 3-butenyl glucosinolate in Brassica crops and its application. Background Art

[0002] Cruciferous crops of Brassica rapa (A genome, 2n = 20) are widely planted in China and have rich variation types, which are collectively referred to as Brassica crops, and are divided into multiple subspecies such as Chinese cabbage, pakchoi, flowering Chinese cabbage, turnip, and oilseed rape. The dominant aliphatic 3-butenyl glucosinolate (Gluconapin, GNA) in Brassica crops can endow them with a special pungent taste, thus seriously affecting the taste and flavor quality of Brassica crops. Therefore, improving the glucosinolate components of Brassica crops or cultivating Brassica crops with low glucosinolate content has important practical significance. At present, traditional breeding methods are used to improve or screen Brassica crops with low glucosinolate content, which have problems such as long cycle, low efficiency, and time-consuming and laborious. With the development and application of molecular marker technology, it shows great advantages in crop cultivation and can overcome many of the above problems. Therefore, it is of great significance to explore a molecular marker that can be applied to improve or screen Brassica crops with low glucosinolate content. Summary of the Invention

[0003] The object of the present invention is to provide an SSR molecular marker related to the content of 3-butenyl glucosinolate in Brassica crops and its application to solve the problems existing in the above-mentioned prior art. The present invention finds that the markers GLS47-8 and GLS34-12 are closely linked to the target traits and can accurately be used to judge the content of 3-butenyl glucosinolate in Brassica crops, and can be used as molecular markers for screening Brassica crops with low glucosinolate content, which is of great significance for the breeding of Brassica crops with low glucosinolate content.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides an SSR molecular marker related to the content of 3-butenyl glucosinolate in Brassica crops. The numbers of the SSR molecular markers are GLS47-8 and GLS34-12. The GLS47-8 and the GLS34-12 are located at 3.348-3.362 Mb on chromosome A01 of the Brassica genome, and the physical distance is 140 kb. The accession number of the Brassica genome is Bra011201 (http: / / brassicadb.org / brad / ).

[0006] Preferably, the GLS47-8 is amplified by using the primer pair shown in SEQ ID NO: 1-2, and the GLS34-12 is amplified by using the primer pair shown in SEQ ID NO: 3-4.

[0007] The present invention also provides a primer set for amplifying the SSR molecular marker, and the primer set is as shown in SEQ ID NO: 1-4.

[0008] The present invention also provides a kit for detecting a molecular marker linked to the content of 3-butenyl glucosinolate in brassica crops, including a primer set for amplifying the SSR molecular marker, and the primer set is as shown in SEQ ID NO: 1-4.

[0009] The present invention also provides the application of the SSR molecular marker, or the primer set, or the kit:

[0010] (1) Application in screening brassica crop varieties or lines with low 3-butenyl glucosinolate content;

[0011] (2) Application in breeding brassica crops with low 3-butenyl glucosinolate content;

[0012] (3) Application in improving the content of 3-butenyl glucosinolate in brassica crops.

[0013] Preferably, using the SSR molecular marker to label the brassica crops, in the lines with homozygous high 3-butenyl glucosinolate genotype, the phenotype of the lines shows a high content of 3-butenyl glucosinolate; in the lines with low 3-butenyl glucosinolate genotype, the phenotype of the lines shows a low content of 3-butenyl glucosinolate.

[0014] Preferably, the brassica crops include Chinese cabbage and turnip.

[0015] The present invention will improve the application of the SSR molecular marker, or the primer set, or the kit in the genetic diversity analysis and genetic map construction of brassica crops.

[0016] Preferably, the brassica crops include Chinese cabbage and turnip.

[0017] The present invention discloses the following technical effects:

[0018] Previously, the inventor has screened 'A01-5' with high GNA content, which is a chromosome segment substitution line created by using 'ECD04' and '59-1'. The present invention uses the parents 'A01-5' and '59-1' to construct an F 2 secondary population, identify the genotypes of the population, and find that the F 2 secondary population shows segregation. Collect the leaves of the individual plants in the population to extract glucosinolates for phenotypic identification, and the GNA content shows a normal distribution in the F 2 secondary population, which conforms to the characteristics of quantitative trait inheritance. Combining with the F 2The phenotypes and genotypes of individual plants in the secondary population were analyzed to identify multiple recombinant types, and finally the mapping interval was narrowed down to between markers GLS47-8 and GLS34-12, namely 3.348-3.362 Mb on chromosome A01, with a physical distance of 140 kb in this interval.

[0019] The present invention also found through the verification of experimental materials that GLS47-8 and GLS34-12 can accurately identify Brassica crops with different GNA contents, providing new molecular markers for screening Brassica crop varieties or lines with different GNA contents, and having important guiding significance for improving the glucosinolate components of Brassica crops and applying them to low-glucosinolate breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the polyacrylamide gel electrophoresis gel diagram of primers GLS47-8 and GLS34-12; Note: P1: Parent 'A01-5', P2: Parent '59-1'; The remaining lanes are individual plants numbered 1-100 in the F 2 population; A, B are the electrophoresis diagrams amplified by primer GLS47-8, and C, D are the electrophoresis diagrams amplified by primer GLS34-12;

[0022] Figure 2 It is the frequency distribution diagram of GNA content in the F 2 population;

[0023] Figure 3 It is the fine mapping of the gene controlling GNA content and the map analysis of recombinant types; Note: The white columns are low-glucosinolate genotypes, the gray columns are heterozygous genotypes, and the black columns are high-glucosinolate genotypes; The letter 'T' represents the high-glucosinolate phenotype, and the letter 'L' represents the low-glucosinolate phenotype;

[0024] Figure 4 It is the distribution diagram of GNA content of parents and other Brassica crop germplasm resources; Chinese cabbage is the germplasm resources of 1-25 Chinese cabbages; Turnip is the germplasm resources of 26-50 turnips;

[0025] Figure 5Verification of molecular markers GLS47-8(A) and GLS34-12(B) in germplasm resource materials of Brassica rapa L. ssp. pekinensis; Note: P1: Parent 'A01-5', P2: Parent '59-1'; The plants numbered 1-25 are germplasm resources of Chinese cabbage with low GNA glucosinolate, and the plants numbered 26-50 are germplasm resource materials of turnip with high GNA glucosinolate. Detailed implementation mode

[0026] The various exemplary implementation modes of the present invention will be described in detail below. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation modes of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are meant to include but not limited to.

[0031] Previously, the inventors used the Chinese cabbage inbred line '59-1' with low GNA content and the turnip 'ECD04' with high GNA content as parents respectively, and completed multiple generations of backcrossing and self-crossing by using SSR marker-assisted selection technology, and successfully screened out the chromosome segment substitution line 'A01-5' strain with significantly increased GNA content (see the literature "Fine mapping of the glucosinolate synthesis gene qGSL1 in Chinese cabbage using chromosome segment substitution lines" for details). Using 'A01-5' and '59-1' as parents to construct an F 2 derived population and perform BSA sequencing, and successfully located the locus controlling GNA synthesis within the interval named qGSL1.1 on chromosome A01, in the interval of 3.3-3.7 Mb on chromosome A01. A gene (Bra011201) highly homologous to the Arabidopsis thaliana glucosinolate synthesis gene AtGGP1 was found in this interval and named BrGGP1.

[0032] Preparation of some reagents involved in the following examples:

[0033] (1) 10×TBE solution (1 L): Weigh 108 g of Tris, 58 g of boric acid, and 8.5 g of EDTA respectively, and make up to 1 L with sterile distilled water;

[0034] (2) Ammonium persulfate (APS) solution: The mass-volume ratio of APS powder to distilled water is 1:10;

[0035] (3) Affinity silane solution (500 mL): Add 25 mL of glacial acetic acid, 450 mL of absolute ethanol, and 25 mL of affinity silane respectively, mix well and set aside;

[0036] (4) Bromophenol blue solution (100 mL): Add 98 mL of deionized formamide, 0.1 g of bromophenol blue powder, 0.1 g of xylene cyanol, and 2 mL of 0.5 M disodium ethylenediaminetetraacetate (EDTA-2Na, pH = 8.0) respectively, and make up to 100 mL with sterile distilled water;

[0037] (5) Polyacrylamide gel solution (1 L): Add 420 g of urea, 527 g of acrylamide, 3 g of N,N'-methylenebisacrylamide, and 100 mL of 10×TBE respectively, make up to 1 L with sterile distilled water, and heat and dissolve in a water bath at 40-60 °C;

[0038] (6) CTAB solution (200 ml): Add 20 mL of 1 mol / L Tris-HCl (pH = 8.0), 8 mL of 0.5 mol / L EDTA, 56 mL of 5 mol / L NaCl, and 4 g of CTAB powder respectively, make up to 200 mL with distilled water, sterilize at 120 °C for 40 min, and after sterilization, let it stand at room temperature for several minutes and then add 0.4% β-mercaptoethanol (2% CTAB lysis buffer = 1:250);

[0039] (7) Dextran solution: Add 1 g of Sephadex (dextran) powder and 25 mL of formic acid into a 50 mL centrifuge tube, let it stand horizontally for 24 h. After 24 h, discard the supernatant, add formic acid again to 10 mL, and use it while shaking.

[0040] (8) Preparation of sulfatase solution

[0041] a: Pour 0.15 g of sulfatase powder after weighing into a 50 mL centrifuge tube, and add 2 mL of 40% ethanol (volume ratio of absolute ethanol to sterilized water = 2:3), shake slowly for 20 s;

[0042] b: Put it into a centrifuge, set the centrifuge at 8000 rpm, 4 °C, and centrifuge for 10 min;

[0043] c: Pour the supernatant obtained after centrifugation into a new centrifuge tube, and add absolute ethanol to the 2 mL scale line, shake slowly for 20 s;

[0044] d: Put it into a centrifuge, set the centrifuge at 8000 rpm, 4 °C, and centrifuge for 10 min;

[0045] e: Discard the supernatant solution, add 10 mL of sterilized water, vortex until the precipitate disappears and the solution is thoroughly mixed;

[0046] f: Aliquot with a 2 mL centrifuge tube and store at -80 °C in the refrigerator for later use. Dilute 10 times before use.

[0047] Example 1

[0048] 1. Test materials

[0049] In this experiment, the chromosome segment substitution line 'A01-5' with high GNA content and the Chinese cabbage inbred line '59-1' with low GNA content were used as parents to construct an F 2 segregating population of 245 individual plants, which were planted in the Back Mountain Herbal Garden Base of Shenyang Agricultural University.

[0050] 2. Identification of genotypes

[0051] 2.1 DNA extraction

[0052] Extract DNA by the CTAB method according to the conventional method steps.

[0053] 2.2 Design of SSR linkage markers

[0054] To further narrow down the qGSL1.1 mapping interval and develop molecular markers tightly linked to GNA content, log in to the Chinese cabbage website (http: / lbrassicadb.org / brad / ) to download the sequence of the 3.3 - 3.7 Mb interval on chromosome A01 where qGSL1.1 is located. Use the SSRHunter software to find the potential SSR loci within the interval and select sequences with 3 - 5 single - base repeats. Use the Primer5.0 software to design denser markers. The criteria for selecting forward and reverse single - strand primers during the design process are as follows: the length of the single - strand primer ranges from 18 - 24 bp, the annealing temperature ranges from 45 - 60 °C and the difference in annealing temperature between the forward and reverse primers should not be greater than 1 °C, the GC% ranges from 40 - 50%, and the length of the PCR product ranges from 100 - 200 bp.

[0055] 2.3 PCR Reaction System and Procedure

[0056] Dilute the primer stock solution 10 - fold for PCR amplification experiments. For polyacrylamide gel electrophoresis experiments, use a 10 μL PCR reaction system as shown in Table 1 below:

[0057] Table 1 PCR Reaction System

[0058] Reagent Dosage DNA 2 μL PCR Forward Primer 0.5 μL PCR Reverse Primer 0.5 μL 2×Taq Master Mix 5 μL <![CDATA[ddH 2 O]]> 2 μL

[0059] The PCR reaction procedure is shown in Table 2 below:

[0060] Table 2 PCR Reaction Procedure

[0061] Step Temperature Time Pre-denaturation 95℃ 2 min Denaturation 94℃ 30s Annealing 54-60℃ 30s Extension 72℃ 30s Final extension 72℃ 5 min

[0062] The process from denaturation to extension is cycled 35 - 40 times, and the annealing temperature is determined according to the lowest annealing temperature of the forward and reverse primers on the primer report form. The PCR products are placed in a 4 °C refrigerator and can be stored for up to 7 days.

[0063] 2.4 Polyacrylamide Gel Electrophoresis

[0064] Use polyacrylamide gel solution for electrophoresis detection.

[0065] 2.5 Detection of GNA Content

[0066] 2.5.1 Extraction of Glucosinolates

[0067] (1) Take fresh leaves of individual plants in the population at 40 d of the growth period, put them in a - 80 °C freezer for storage. Put the sample leaves into a self - sealing bag, open the bag mouth and label them. Use a freeze - dryer to treat them at - 110 °C for 48 h. After taking them out, knead the self - sealing bag repeatedly by hand until it becomes a powder state. Weigh 0.1 g of the sample using an analytical balance, put it into a 2 mL centrifuge tube, make 3 replicates for each sample, label them and then put them in a - 80 °C refrigerator for storage;

[0068] (2) Prepare the dextran solution one day in advance; preheat 70% methanol in a 70 °C water bath; prepare a 0.1 mol / L sinigrin standard sample using sinigrin powder and sterilized water.

[0069] (3) Take out the sample powder from -80 °C, add 1 mL of 70% methanol to each tube, and vortex to thoroughly mix the powder and the solution.

[0070] (4) Place the centrifuge tube in a 70 °C water bath for heat treatment for 10 min.

[0071] (5) After taking out, set the centrifuge to 17000 rpm, 4 °C, and centrifuge for 8 min.

[0072] (6) Aspirate the supernatant and transfer it to a new 15 mL centrifuge tube.

[0073] (7) Repeat steps (3) - (6) three times, collect the supernatant solutions from the three times, set the centrifuge to 2500 rpm, 4 °C, and centrifuge for 10 min.

[0074] (8) Prepare a clean short glass Pasteur pipette, stuff fine glass wool under the thin part of the short glass Pasteur pipette, add 2 mL of sterilized water to wash the glass wool, and then add 300 μL of dextran solution and 1.5 mL of formic acid respectively.

[0075] (9) Transfer the supernatant obtained after centrifugation into the pipette, and then add 1 mL of sterilized water.

[0076] (10) Insert the short glass Pasteur pipette into the rubber tube (insert the rubber tube into the operation board in advance, and use a hot glue gun to melt the glue to completely seal the bottom of the rubber tube).

[0077] (11) Add 250 μL of the prepared phosphatase solution.

[0078] (12) Place the operation board in a clean and well-ventilated place and let it stand overnight for 16 - 18 h.

[0079] (13) The next day, add 1.5 mL of sterilized water to the pipette for collection, then filter with a 0.22 μm water-based filter head and transfer it to a brown sample bottle.

[0080] (14) Perform the detection in a timely manner. If the detection cannot be carried out in a timely manner, it can be placed in a 4 °C refrigerator and can be stored for up to 3 - 5 days.

[0081] 2.5.2 Detection by high performance liquid chromatography

[0082] The glucosinolate components of the sample material were separated and their contents were detected using a high-performance liquid chromatograph. By combining the distribution coefficients of each component, the corresponding retention time nodes were found, their peaks were calculated, and the components and contents of the glucosinolates were analyzed. This method is convenient and fast, and the chromatogram of the detection process can be directly and clearly obtained.

[0083] The operation method is as follows: To ensure the accuracy of the detected peak area and better determine the components and contents of the glucosinolates, a balance test of the high-performance liquid chromatograph must be carried out before the experiment, and the instrument is subjected to an empty treatment test for 30 minutes. The special brown vial of the high-performance liquid chromatograph containing the solution after extracting the glucosinolates is placed into the high-performance liquid chromatograph and arranged in the order of the labels; the equipment coefficients of the detection process are adjusted, and after calibrating the instrument, the detection is carried out at an absorbance value of 229 nM, and the detection time for each tube of sample is 30 minutes; first, the 30-minute balance baseline is calibrated, and after accurate balancing, the experiment is carried out; in the first step, the standard sample Sinigrin is first detected by high-performance liquid chromatography, and then the high-performance liquid chromatography detection of the population material is carried out.

[0084] Table 3 Parameter settings of the high-performance liquid chromatograph

[0085] Parameter Condition Mobile phase <![CDATA[H 2 O, CH 3 OH, C 2 H 3 N]]> Temperature 30±5℃ Volume 10 mL UV wavelength 229 nm

[0086] Before using the high-performance liquid chromatograph to detect the sample, to ensure the accuracy of the peak area, the parameters of the gradient elution mobile phase required for the experiment must be set. The calculation formula of the GNA content refers to Ma Siwen's "Fine mapping of the glucosinolate synthesis gene qGSL1 in Chinese cabbage using chromosome segment substitution lines". The GSL concentration unit is expressed in μmol / g, and the molecular weight of GNA is 373.4.

[0087] 2.6 Precise mapping of the qGSL1.1 locus and development of tightly linked markers

[0088] SSR molecular markers were designed according to the genomic sequence of the physical position of qGSL1.1 on the chromosome. First, the designed SSR encrypted markers were screened for polymorphism between the parents 'A01-5' and '59-1', and then the markers with polymorphism between the parents were used to identify the genotypes of the F 2 population individual plants. The recombination rate was calculated using JoinMap 4 software and a genetic linkage map was constructed. The LOD value was calculated using the software QTL IciMapping, and a specific critical value was manually set, and then the QTL mapping-linked loci were inferred to complete the analysis of the QTL loci and determine the tightly linked SSR molecular markers.

[0089] 3. Results and analysis

[0090] 3.1 Genotype identification and analysis of the segregating population

[0091] In order to further narrow down the mapping interval of qGSL1.1 for fine mapping and obtain more closely linked markers, the present invention uses Primer5.0 software to design multiple pairs of SSR encryption markers for the mapping interval of 3.3 - 3.7 Mb on chromosome A01 where qGSL1.1 is located, so as to narrow down the mapping interval. A total of 36 pairs of SSR markers are designed. After screening, it is found that 11 pairs of encryption markers among them have significant polymorphisms between the parents (Table 4). Some polyacrylamide gel electrophoresis gel diagrams are as Figure 1 shown. Furthermore, these 11 pairs of SSR markers are used to identify and record the genotypes of individual plants in the F 2 population. The results show that obvious segregation phenomenon appears in the F 2 population.

[0092] Table 4 SSR Polymorphic Primer Sequences

[0093]

[0094]

[0095] 3.2 Detection and Analysis of GNA Content in F 2 Segregation Population

[0096] The components and contents of glucosinolates in the F 2 population are detected and analyzed by high performance liquid chromatography. By observing the chromatogram and analyzing the different peaks formed at different time nodes, the relevant calculations of different glucosinolate components and their contents are determined.

[0097] Using SPSS statistical analysis software, the phenotypic trait data of the GNA content of the parents '59 - 1', 'ECD04', 'A01 - 5' and individual plants in the F 2 population are statistically analyzed. The results show that, as Figure 2 shown, the GNA content shows a normal distribution in the F 2 population, presenting a significant single - peak curve, indicating that this trait belongs to a typical quantitative trait controlled by multiple genes and can be used for the next QTL experimental analysis. And in the F 2 population, there are some plant lines with GNA content significantly higher and lower than that of the parents, showing a significant transgressive segregation phenomenon, which further illustrates that this genetic mode is quantitative trait inheritance and has the characteristics of continuous inheritance. Among them, the GNA contents of '59 - 1', 'ECD04' and 'A01 - 5' marked by red arrows are distributed in the extremely low and extremely high content regions respectively.

[0098] 3.3 Fine Mapping Analysis of qGSL1.1 Gene

[0099] Perform QTL genetic map analysis on 11 pairs of SSR encryption markers with polymorphisms, combined with F 2The glucosinolate content phenotypes and genotypes of 245 individual plants in the population were used to classify the recombinant types. By analyzing the genotypes of the recombinants under different markers and combining their phenotypes, as Figure 3 shown, a total of 6 representative recombinant types were identified. The present invention found that the lines with homozygous high-glucosinolate genotypes under the markers GLS47-8 and GLS34-12 also showed higher GNA content in their phenotypes, while the lines identified with low-glucosinolate genotypes showed significantly lower GNA content. Therefore, the present invention finally finely mapped the key locus qGSL1.1 controlling GNA synthesis between the markers GLS47-8 and GLS34-12, located at 3.348-3.362 Mb on chromosome A01, and the physical distance of this interval is 140 kb, laying a foundation for further functional analysis of candidate genes.

[0100] Example 2 Verification of tightly linked markers in other Brassica rapa crops

[0101] The present invention selected 50 Brassica rapa crops, including 25 Chinese cabbages and 25 turnip inbred lines (see Table 5), as Figure 4 shown. The average GNA content of 25 Chinese cabbage inbred lines was 0.25 μmol / g, and the average GNA content of 25 turnip inbred lines was 22.5 μmol / g. As Figure 5 shown, the markers GLS47-8 and GLS34-12 can clearly distinguish Brassica rapa crop materials with low and high GNA contents.

[0102] Table 5 Information on 50 germplasm resources of Brassica rapa crops

[0103] Material label Name of Chinese cabbage germplasm resource Material label Name of turnip germplasm resource 1 252 26 CGN1 2 Chiifu 27 CGN2 3 BJN3-2 28 CGN3 4 11379 29 CGN4 5 026 30 CGN5 6 11420 31 CGN6 7 11390 32 CGN7 8 11377 33 CGN8 9 85-74 34 CGN9 10 11396 35 CGN10 11 11473 36 CGN11 12 11475 37 CGN12 13 11389 38 CGN13 14 12A 39 CGN14 15 Shinki 40 CGN15 16 RI-203 41 CGN16 17 18A18 42 CGN17 18 AST474 43 CGN18 19 AST2134 44 CGN19 20 RI-9 45 CGN20 21 AST552 46 CGN21 22 JBR-2 47 CGN22 23 JBR-3 48 CGN23 24 PZ20 49 CGN24 25 222 50 CGN25

[0104] Note: For the Chinese cabbage germplasm resources numbered 1-25, refer to the paper "Mapping and cloning of the clubroot resistance gene CRd in Chinese cabbage" (Fu Pengyu, 2019); for the turnip germplasm resources numbered 26-50, refer to the paper "Identification of clubroot resistance and analysis of CR gene-linked markers in turnip germplasm" (Huang Xinbiao, 2022).

[0105] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An SSR molecular marker related to the content of 3-butenyl glucosinolate in cabbage crops, characterized in that: The SSR molecular markers are numbered as GLS47-8 and GLS34-12, and the GLS47-8 and GLS34-12 are located at 3.348-3.362 Mb on the A01 chromosome of the cabbage genome, with a physical distance of 140 kb, and the accession number of the cabbage genome is Bra011201; The GLS47-8 was amplified using the primer pair shown in SEQ ID NO: 1-2, and the GLS34-12 was amplified using the primer pair shown in SEQ ID NO: 3-4.

2. Use of the reagent for amplifying the SSR molecular marker according to claim 1 in any of the following: (1) Application in screening cabbage crop varieties or lines with low 3-butenyl glucosinolate content; (2) Application in the breeding of cabbage crops with low 3-butenyl glucosinolate content; (3) Application in identifying the content of 3-butenyl glucosinolate in cabbage crops; Amplifying the cabbage crop using the primer pair of claim 1, if the cabbage crop has the same amplification result as 'A01-5', the cabbage crop shows a high 3-butenyl glucosinolate content; if the cabbage crop has the same amplification result as '59-1', the cabbage crop shows a low 3-butenyl glucosinolate content; The cabbage crops are Chinese cabbage and turnip.

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