Mustard KASP molecular marker combination and application thereof in germplasm resource identification and breeding

Abstract

The invention discloses a brassica juncea KASP molecular marker combination and application thereof in germplasm resource identification and breeding, and belongs to the technical field of molecular markers. The mustard KASP molecular marker set is developed from 193 mustard germplasm resources based on whole genome re-sequencing, the mustard KASP molecular marker can quickly, accurately and effectively identify and evaluate germplasm resource materials or bred varieties, and the problems of resource redundancy, inconvenient storage and management, intellectual property disputes and the like caused by accumulation of a large amount of germplasm can be solved. The molecular marker is provided for genetic map construction, gene positioning and molecular assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to a KASP molecular marker combination for mustard and its application in germplasm resource identification and breeding. Background Technology

[0002] Mustard greens (Brassica juncea (L.) Czern.), an annual herbaceous plant belonging to the Brassicaceae family and the Brassica genus, are a major vegetable consumed in my country.

[0003] Molecular markers (RAPD, SSR, Indel, SNP, etc.) are widely used in the evaluation of genetic diversity of mustard germplasm resources, genetic mapping and gene localization, and marker-assisted breeding. Among these, RAPD technology is relatively susceptible to various factors. Whether it's the quality and concentration of the template, short primer sequences, the number of PCR cycles, the complexity of genomic DNA, or technical equipment, all can lead to unstable and difficult-to-reproduce RAPD results. SSR markers, due to their high polymorphism and ease of operation, were once widely used in vegetable crop genetic breeding in my country. However, they cannot meet the needs of large-scale, high-throughput, and automated detection. SNP markers, as a third-generation molecular marker technology, have the following advantages: first, high density and more uniform distribution in the genome; second, high throughput, with detection sites reaching millions; and third, ease of automated data statistics and data integration and comparison. Among these, competitive allele-specific PCR (KASP) is a genotyping technique based on fluorescence detection. This technology is now mainly used in SNP or Indel gene typing studies and is gradually becoming a major technical means for genetic map construction, gene localization, molecular-assisted breeding, and germplasm resource evaluation. Summary of the Invention

[0004] The purpose of this invention is to provide a KASP molecular marker combination for mustard and its application in germplasm resource identification and breeding, thereby solving the problems existing in the prior art. This invention utilizes core KASP molecular markers for mustard developed based on whole-genome resequencing, providing a new approach for mustard genetic mapping, gene localization, and molecular-assisted breeding.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a KASP molecular marker combinatorial for constructing a mustard fingerprint spectrum, which consists of KASPJC2, KASPJC4~10, KASPJC12~14, KASPJC17~18, KASPJC22~27, KASPJC31 and KASPJC33; KASPJC2 is located on chromosome AA01 of the mustard genome, and there is a base polymorphism A / G at position 21508882; KASPJC4 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism G / A at position 9149701; KASPJC5 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism T / A at position 25375102; KASPJC6 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 20858767; KASPJC7 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 1207962; KASPJC8 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism T / G at position 33425368; KASPJC9 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / T at position 4179480; KASPJC10 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / G at position 10059237; KASPJC12 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism A / C at position 10191874; KASPJC13 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism G / A at position 10553690; KASPJC14 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism T / G at position 16488896; KASPJC17 is located on chromosome AA09 of the mustard genome, and there is a base polymorphism G / T at position 52646724; KASPJC18 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism T / G at position 7218713; KASPJC22 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism A / T at position 46667455; KASPJC23 is located on chromosome BB02 of the mustard genome, and has a base polymorphism G / A at position 44834278; KASPJC24 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism C / G at position 11524175; KASPJC25 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism T / C at position 9110022; KASPJC26 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism A / G at position 1462186; KASPJC27 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism G / T at position 1462170; KASPJC31 is located on chromosome BB07 of the mustard genome, and there is a base polymorphism T / A at position 12663845; KASPJC33 is located on chromosome BB07 of the mustard genome, and has a base polymorphism C / G at position 28738996.

[0006] The second technical solution of the present invention is a primer set for specifically detecting the KASP molecular marker combination, which consists of primers as shown in SEQ ID NO. 4~6, SEQ ID NO. 10~30, SEQ ID NO. 34~42, SEQ ID NO. 49~54, SEQ ID NO. 64~81, SEQ ID NO. 91~93 and SEQ ID NO. 97~99.

[0007] The third technical solution of the present invention is a reagent, kit, or chip for specifically detecting the KASP molecular marker combination, including the primer set.

[0008] The fourth technical solution of the present invention is the application of the KASP molecular marker combination in the identification of mustard germplasm resources or variety identification.

[0009] The fifth technical solution of the present invention is the application of the KASP molecular marker combination in constructing a genetic map of mustard.

[0010] The sixth technical solution of this invention is the application of the KASP molecular marker combination in mustard gene localization or molecular marker-assisted breeding.

[0011] The seventh technical solution of this invention is a method for constructing a mustard fingerprint spectrum, comprising the following steps: (1) Collect tissue samples and extract total DNA from the samples; (2) PCR amplification was performed using the primer set, and the amplification products were sequenced; (3) Identify the genotype of the site where the KASP molecular marker combination is located on the sample genome, and construct a mustard fingerprint map using the identified genotype.

[0012] The eighth technical solution of the present invention is a method for identifying mustard varieties, comprising the following steps: (1) Collect tissue samples from the samples to be identified and extract total DNA from the samples; (2) Identify the genotype of the site where the KASP molecular marker combination is located on the genome of the sample to be identified; (3) Compare the individual genotype of the sample to be identified with the constructed fingerprint map, and determine the variety of the sample to be identified based on the comparison results.

[0013] The ninth technical solution of the present invention is the application of the KASP molecular marker combination, the primer set, and the reagents, kits, or chips in the identification of mustard varieties.

[0014] Based on the above technical solution, the present invention has the following technical effects: This invention develops a core SNP molecular marker for mustard based on KASP technology. This mustard SNP molecular marker can rapidly, accurately, and effectively identify and evaluate germplasm resources or bred varieties, solving problems such as resource redundancy, inconvenient preservation and management, and intellectual property disputes caused by the accumulation of large amounts of germplasm. It provides molecular markers for genetic mapping, gene localization, and molecular-assisted breeding. Attached Figure Description

[0015] Figure 1 Polymorphism map of AA01:KASPJC2 marker in 193 mustard germplasm resources.

[0016] Figure 2 Polymorphism map of AA08:KASPJC18 marker in 193 mustard germplasm resources.

[0017] Figure 3 Genetic distance clustering diagram of 193 mustard accessions based on KASP markers.

[0018] Figure 4 Fingerprint profiles of 28 mustard varieties (lines) Detailed Implementation

[0019] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0020] This invention provides a KASP molecular marker combinatorial array for constructing a mustard fingerprint, consisting of KASPJC2, KASPJC4~10, KASPJC12~14, KASPJC17~18, KASPJC22~27, KASPJC31 and KASPJC33; KASPJC2 is located on chromosome AA01 of the mustard genome, and there is a base polymorphism A / G at position 21508882; KASPJC4 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism G / A at position 9149701; KASPJC5 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism T / A at position 25375102; KASPJC6 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 20858767; KASPJC7 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 1207962; KASPJC8 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism T / G at position 33425368; KASPJC9 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / T at position 4179480; KASPJC10 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / G at position 10059237; KASPJC12 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism A / C at position 10191874; KASPJC13 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism G / A at position 10553690; KASPJC14 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism T / G at position 16488896; KASPJC17 is located on chromosome AA09 of the mustard genome, and there is a base polymorphism G / T at position 52646724; KASPJC18 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism T / G at position 7218713; KASPJC22 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism A / T at position 46667455; KASPJC23 is located on chromosome BB02 of the mustard genome, and has a base polymorphism G / A at position 44834278; KASPJC24 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism C / G at position 11524175; KASPJC25 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism T / C at position 9110022; KASPJC26 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism A / G at position 1462186; KASPJC27 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism G / T at position 1462170; KASPJC31 is located on chromosome BB07 of the mustard genome, and there is a base polymorphism T / A at position 12663845; KASPJC33 is located on chromosome BB07 of the mustard genome, and has a base polymorphism C / G at position 28738996.

[0021] This invention also provides a primer set for specifically detecting the KASP molecular marker combination, consisting of primers as shown in SEQ ID NO. 4~6, SEQ ID NO. 10~30, SEQ ID NO. 34~42, SEQ ID NO. 49~54, SEQ ID NO. 64~81, SEQ ID NO. 91~93 and SEQ ID NO. 97~99.

[0022] Embodiments of the present invention also provide reagents, kits, or chips for the specific detection of the KASP molecular marker combination, including the primer set.

[0023] This invention also provides the application of the KASP molecular marker combination in the identification of mustard germplasm resources or variety identification.

[0024] This invention also provides the application of the KASP molecular marker combination in constructing a genetic map of mustard.

[0025] This invention also provides the application of the KASP molecular marker combination in mustard gene mapping or molecular marker-assisted breeding.

[0026] This invention also provides a method for constructing a mustard fingerprint, comprising the following steps: (1) Collect tissue samples and extract total DNA from the samples; (2) PCR amplification was performed using the primer set, and the amplification products were sequenced; (3) Identify the genotype of the site where the KASP molecular marker combination is located on the sample genome, and construct a mustard fingerprint map using the identified genotype.

[0027] This invention also provides a method for identifying mustard varieties, comprising the following steps: (1) Collect tissue samples from the samples to be identified and extract total DNA from the samples; (2) Identify the genotype of the site where the KASP molecular marker combination is located on the genome of the sample to be identified; (3) Compare the individual genotype of the sample to be identified with the constructed fingerprint map, and determine the variety of the sample to be identified based on the comparison results.

[0028] This invention also provides the application of the KASP molecular marker combination, the primer set, and the reagents, kits, or chips in the identification of mustard varieties.

[0029] The specific methods for the above applications include the following steps: S1. Extract genomic DNA from mustard green samples; S2. Using the DNA from step S1 as a template, PCR amplification was performed using the KASP primer set, and the PCR amplification product was placed on the Bio-Rad CFX Connect real-time fluorescence quantitative PCR instrument to obtain the corresponding product fluorescence signal value and complete the genotyping. S3. The experimental results of step S2 were analyzed using the data analysis software Bio-Rad CFX Manager 3.1.

[0030] Furthermore, the PCR amplification reaction system was 10 µL, including 2 µL of DNA at 20 ng / µL to 30 ng / µL, 0.28 µL of each KASP primer combination (0.07 µL each of the two F primers and 0.14 µL of the R primer), and 5 µL of KASP Mastermixture.

[0031] Furthermore, the PCR amplification reaction conditions were as follows: 94℃, 15 min; 94℃, 20 sec, 63℃~55℃, 1 min, with a decrease of 0.8℃ per cycle, for a total of 10 cycles; 94℃, 20 sec, 55℃, 1 min, for a total of 26 cycles; the fluorescence signal reading conditions were 16℃, 20 s.

[0032] This invention developed a set of KASP molecular markers for mustard from 193 mustard germplasm resources based on whole-genome resequencing. These KASP molecular markers can rapidly, accurately, and effectively identify and evaluate germplasm resources or bred varieties, solving problems such as resource redundancy, inconvenient preservation and management, and intellectual property disputes caused by the accumulation of large amounts of germplasm. They provide molecular markers for genetic mapping, gene localization, and molecular-assisted breeding.

[0033] Example 1 KASP primer design Genomic DNA was extracted from six high-generation inbred lines of mustard, namely J15, J49, J79, J87, J134 and J176.

[0034] Whole-genome resequencing analysis was performed on six high-generation inbred lines of mustard using Illumina HiSeq™ technology (conducted by Guangzhou Gediao Biotechnology Co., Ltd.). The obtained resequencing data were compared with the mustard reference genome. BjuIR: Brassica juncea information resourceT84-66.V2.0 Comparative analysis was performed to select 100 SNP sites that were polymorphic, evenly distributed on the chromosome, and had no other variations within 50 bp before and after the site. Primers were then designed to convert these 100 SNP sites into KASP markers.

[0035] Based on the SNP sites and flanking sequences, KASP marker primers were designed for these differentially expressed sites using Primer Premier 5.0 software, following the primer design method described by Awais et al. (Awais R, WenWE, Gao FM, Zhai SN, Jin H, Liu JD, Guo Q, Zhang YJ, Dreisigacker S, XiaXC, HeZH. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theoretical and Applied Genetics, 2016, 129(10): 1843-1860). KASP molecular markers were developed.

[0036] For each marker, two SNP-specific primers (F1 / F2) and one universal primer (R) were designed. The F1 primer had a specific sequence (5-GAAGGTGACCAAGTTCATGCT-3') added to its tail to bind to FAM fluorescence, and the F2 primer had a specific sequence (5'-GAAGGTCGGAGTCAACGGATT-3') added to its tail to bind to HEX fluorescence. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and a total of 288 pairs of KASP markers were designed.

[0037] Example 2 KASP primer selection (1) 193 mustard germplasm resources were collected to form a natural population of mustard (as shown in Table 1).

[0038] Table 1. Information on DNA materials from mustard greens

[0039] (2) Extraction of DNA from mustard greens: ① Take about 2g of tender mustard leaves, grind them with liquid nitrogen, and quickly add 1000μL of 2% CTAB extraction buffer when the liquid nitrogen is almost evaporated. Mix well and place in a 65℃ water bath for 50min, shaking once every 10min. ② After standing to room temperature, centrifuge at 12000 rpm for 10 min at 4℃, and transfer about 800 μL of supernatant to a new 2 mL centrifuge tube; ③ Add an equal volume of chloroform / isoamyl alcohol (chloroform:isoamyl alcohol = 24:1), invert to mix, let stand for 3-5 minutes, centrifuge at 12000 rpm for 10 minutes at 4℃, and transfer about 600 μL of the supernatant into a new 1.5 mL centrifuge tube; ④ Add an equal volume of anhydrous ethanol pre-cooled at -20℃, mix slowly, invert slowly 20 times, and incubate at -20℃ for 30 min; ⑤ Centrifuge at 12000rpm for 10min at 4℃. After seeing the white precipitate at the bottom, discard the supernatant. Wash the precipitate twice with 800μL of 75% and 95% alcohol respectively. Discard the supernatant and air dry in a fume hood at room temperature. ⑥ Dissolve in 100 μL of sterile water to obtain the DNA of mustard germplasm resources.

[0040] Using six sequencing inbred lines as experimental materials, 100 pairs of developed KASP markers were validated and screened. PCR (polymeric chain reaction) amplification was performed using DNA from J15, J49, J79, J87, J134, and J176 as templates, with the addition of KASP primers and a universal KASP Master mix (purchased from Guangzhou Good Biotech Co., Ltd.).

[0041] The PCR reaction system is as follows: 10 μL of DNA containing 20 ng / μL~30 ng / μL, 1 μL of the molecular marker primers (50 ng / μL). –1 The total volume was 0.5 μL, including 0.1 μL of each of the two F primers, 0.3 μL of the R primer, and 5 μL of 2×KASP Mastermixture.

[0042] The reaction conditions were: 94℃ for 15 min; 94℃ for 20 sec, 61℃~55℃ for 1 min, with a decrease of 0.6℃ per cycle, for a total of 10 cycles; and 94℃ for 20 sec, 55℃ for 1 min, for a total of 26 cycles.

[0043] After the reaction was complete, the PCR amplification products were placed on a Bio-Rad CFX Connect real-time quantitative PCR instrument to obtain the corresponding fluorescence signal values, and genotyping was performed (HEX fluorescence was used to excite one homozygous genotype, FAM fluorescence was used to excite another homozygous genotype, and simultaneous excitation of both FAM and HEX fluorescence was used to excite a heterozygous genotype). The fluorescence signal values ​​were read at 16℃ for 20 seconds, and the experimental results were analyzed using Bio-Rad CFX Manager 3.1 data analysis software.

[0044] Using the successfully genotyped KASP markers, genotyping was performed on 193 mustard germplasm resources, ultimately yielding 34 high-quality KASP markers with clear genotypes. These 34 KASP markers are evenly distributed across the 19 chromosomes of mustard, constituting the core SNP molecular marker set for mustard in this invention. The location information and nucleotide sequence of each marker are shown in Table 2.

[0045] Table 2 Primer information for KASP molecular markers in mustard greens

[0046] Note: A FAM fluorescent tag sequence is added to the 5' end of primer F1, and a HEX fluorescent tag sequence is added to the 5' end of primer F2.

[0047] The allele frequencies and polymorphism information content (PIC) of each marker were calculated using POWER MARKER V3.25 software. The PIC of all KASP markers in 193 germplasm resources ranged from 0.2466 to 0.3750, with an average of 0.3462. Loci with PIC values ​​greater than 0.3462 accounted for 61.7% of all loci. The major allele frequency (MAF) ranged from 0.5 to 0.8255, with an average of 0.6337.

[0048] Example 3 Application of KASP molecular markers in genetic diversity analysis of mustard Genotyping data of 34 KASP-marked mustard natural populations were used to construct an AB matrix. A phylogenetic tree was then built based on the genetic distance matrix using PowerMarker 3.25 software. Neighbor-Joining clustering analysis was performed using MEGA11.0 software. The results are shown in the attached figure. Figure 4 As shown, the 193 mustard germplasm resources can be divided into two major groups: I and II.

[0049] Example 4 Core KASP molecular markers applied to fingerprinting of mustard varieties (lines) From the 34 KASP markers in Example 3, markers with a PIC value greater than the average of 0.3462 and a missing rate of less than 0.1% were selected as core markers for constructing the fingerprint map of mustard varieties (lines). Ultimately, 21 KASP markers were obtained for constructing the fingerprint map of mustard varieties (lines). The core KASP markers are: KASPJC2, KASPJC4, KASPJC5, KASPJC6, KASPJC7, KASPJC8, KASPJC9, KASPJC10, KASPJC12, KASPJC13, KASPJC14, KASPJC17, KASPJC18, KASPJC22, KASPJC23, KASPJC24, KASPJC25, KASPJC26, KASPJC27, KASPJC31, and KASPJC33.

[0050] Genotyping of 28 mustard varieties (lines) (Table 3) was performed using 21 core KASP markers to construct DNA fingerprint profiles for the mustard varieties (lines). The core locus genotyping results of the mustard varieties (lines) were converted into binary coded data to obtain the fingerprint profiles of the mustard varieties (lines). Figure 4 This DNA fingerprint can be used to effectively distinguish mustard varieties (lines).

[0051] Table 3. Details of 28 Mustard Varieties (Lines)

[0052] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A KASP molecular marker combinatorial system for constructing a mustard fingerprint, characterized in that, It consists of KASPJC2, KASPJC4~10, KASPJC12~14, KASPJC17~18, KASPJC22~27, KASPJC31 and KASPJC33; KASPJC2 is located on chromosome AA01 of the mustard genome, and there is a base polymorphism A / G at position 21508882; KASPJC4 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism G / A at position 9149701; KASPJC5 is located on chromosome AA02 of the mustard genome, and there is a base polymorphism T / A at position 25375102; KASPJC6 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 20858767; KASPJC7 is located on chromosome AA03 of the mustard genome, and there is a base polymorphism T / C at position 1207962; KASPJC8 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism T / G at position 33425368; KASPJC9 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / T at position 4179480; KASPJC10 is located on chromosome AA06 of the mustard genome, and there is a base polymorphism A / G at position 10059237; KASPJC12 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism A / C at position 10191874; KASPJC13 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism G / A at position 10553690; KASPJC14 is located on chromosome AA08 of the mustard genome, and there is a base polymorphism T / G at position 16488896; KASPJC17 is located on chromosome AA09 of the mustard genome, and there is a base polymorphism G / T at position 52646724; KASPJC18 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism T / G at position 7218713; KASPJC22 is located on chromosome BB01 of the mustard genome, and there is a base polymorphism A / T at position 46667455; KASPJC23 is located on chromosome BB02 of the mustard genome, and has a base polymorphism G / A at position 44834278; KASPJC24 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism C / G at position 11524175; KASPJC25 is located on chromosome BB04 of the mustard genome, and there is a base polymorphism T / C at position 9110022; KASPJC26 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism A / G at position 1462186; KASPJC27 is located on chromosome BB05 of the mustard genome, and there is a base polymorphism G / T at position 1462170; KASPJC31 is located on chromosome BB07 of the mustard genome, and there is a base polymorphism T / A at position 12663845; KASPJC33 is located on chromosome BB07 of the mustard genome, and exhibits a base polymorphism C / G at position 28738996.

2. A primer set for specific detection of the KASP molecular marker combination of claim 1, characterized in that, It consists of primers as shown in SEQ ID NO.4~6, SEQ ID NO.10~30, SEQ ID NO.34~42, SEQ ID NO.49~54, SEQ ID NO.64~81, SEQ ID NO.91~93 and SEQ ID NO.97~99.

3. A reagent, kit, or chip for specific detection of the KASP molecular marker combination of claim 1, characterized in that, Includes the primer set described in claim 2.

4. The application of the KASP molecular marker combination as described in claim 1 in the identification of mustard germplasm resources or variety identification.

5. The application of the KASP molecular marker combination as described in claim 1 in constructing a genetic map of mustard.

6. The application of the KASP molecular marker combination as described in claim 1 in mustard gene localization or molecular marker-assisted breeding.

7. A method for constructing a fingerprint spectrum of mustard greens, characterized in that, Includes the following steps: (1) Collect tissue samples from the samples and extract total DNA from the samples; (2) Perform PCR amplification using the primer set described in claim 2, and sequence the amplification products; (3) Identify the genotype of the site where the KASP molecular marker combination as described in claim 1 is located on the genome of the sample, and construct a mustard fingerprint map using the identified genotype.

8. A method for identifying mustard varieties, characterized in that, Includes the following steps: (1) Collect tissue samples from the samples to be identified and extract total DNA from the samples; (2) Identify the genotype of the site on the genome of the sample to be identified as described in claim 1; (3) The individual genotype of the sample to be identified is compared with the fingerprint spectrum constructed in claim 7, and the variety of the sample to be identified is determined based on the comparison results.

9. The application of the KASP molecular marker combination of claim 1, the primer set of claim 2, and the reagent, kit, or chip of claim 3 in the identification of mustard varieties.

Images