CN112646917A - SNP molecular marker related to Chinese cabbage flower color characters, detection primer, detection kit and application thereof - Google Patents
SNP molecular marker related to Chinese cabbage flower color characters, detection primer, detection kit and application thereof Download PDFInfo
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Abstract
The invention provides an SNP molecular marker related to Chinese cabbage flower color traits, a detection primer, a detection kit and application thereof, and belongs to the technical field of crop molecular marker assisted breeding. SNP molecular markers related to the Chinese cabbage flower color traits are located in the positions shown as SEQ ID NO: 1, the 101 th base of the gene segment shown in 1 is G/-, the flower color of the Chinese cabbage is yellow flower when the base is G, and the flower color of the Chinese cabbage is white flower when the base is absent. The primers for detecting the colors of the Chinese cabbages based on the KASP technology comprise a reverse primer and two forward primers of which the 5' end sequences are label sequences marked with FAM or HEX fluorescent groups. Because the SNP molecular marker and the primer thereof can accurately identify the flower color character of the Chinese cabbage, the invention provides the application of the SNP molecular marker, the primer or the kit in the detection of the flower color of the Chinese cabbage or the breeding of the Chinese cabbage plant, which is not limited by environmental factors, can greatly reduce the workload and is beneficial to the acceleration of the breeding of the Chinese cabbage.
Description
Technical Field
The invention belongs to the technical field of crop molecular marker assisted breeding, and particularly relates to an SNP molecular marker related to Chinese cabbage flower color traits, and a detection primer, a detection kit and application thereof.
Background
The flower color is one of the phenotypic characters of the plant adaptive to the environment, is an important visual signal for inducing insects to pollinate by insect-vector plants, and has important significance for the survival and the propagation of the plants. Meanwhile, the flower color can influence the aphid to feed, the crop varieties with different flower colors are bred in production, the aphid disease transmission effect is reduced, the energy balance of petals can be maintained, and the damage to flower organs is avoided. The flower color can be used for identifying true and false hybrid species in production practice.
The Chinese cabbage is one of the vegetable crops with the largest cultivation area and yield in China, and has important economic value. In brassica, the white flower character is more researched in brassica napus and brassica juncea and less researched in Chinese cabbage. At present, no molecular marker related to the identification of the white flower character of the Chinese cabbage is reported.
Disclosure of Invention
In view of the above, the present invention provides an SNP molecular marker related to the flower color traits of Chinese cabbage, and the SNP molecular marker can accurately distinguish the flower color and white flower color phenotypes of Chinese cabbage.
The invention also aims to provide a detection primer, a detection kit and application for identifying the color traits of the Chinese cabbage.
The invention provides an SNP molecular marker related to Chinese cabbage flower color traits, which is positioned in a sequence shown as SEQ ID NO: 1, wherein-represents deletion, and the 101 th base of the gene fragment shown in 1 is G/-.
The invention provides a primer for detecting Chinese cabbage flower color based on competitive allele specific PCR technology, which comprises TXBH83Fa, TXBH83Fb and TXBH 83R;
the nucleotide sequence of the TXBH83Fa is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the TXBH83Fb is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the TXBH83R is shown as SEQ ID NO: 4 is shown in the specification;
wherein the 5' end sequence of the TXBH83Fa or TXBH83Fb is a tag sequence labeled with FAM or HEX fluorescent group.
The invention provides a kit for detecting the flower color of Chinese cabbage based on competitive allele specificity PCR technology, which comprises the primer.
Preferably, the kit further comprises KASP Master mix.
The invention provides application of the SNP molecular marker, the primer or the kit in detecting the flower color of the Chinese cabbage or detecting the genotype of the flower color of the Chinese cabbage.
The invention provides application of the SNP molecular marker, the primer or the kit in Chinese cabbage plant breeding.
The invention provides a method for detecting the flower color of Chinese cabbage in high flux, which comprises the following steps:
1) extracting the Chinese cabbage genome DNA of the flower color to be detected;
2) using the genomic DNA in the step 1) as a template, and performing competitive allele specific PCR amplification by using the primer to obtain a KASP amplification product;
3) reading the fluorescence signal of the KASP amplification product by an end point method to obtain a genotyping result;
the genotype fragment obtained by the amplification of the primers TXBH83Fa and TXBH83R is from Chinese cabbage with yellow flower character;
the genotype fragments obtained by the amplification of the primers TXBH83Fb and TXBH83R are from Chinese cabbage with white flower character.
Preferably, the reaction procedure for the competitive allele-specific PCR amplification is: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s, and comprises 10 cycles, and the cycle of the second stage is started, wherein each cycle is reduced by 0.6 ℃; in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; fourth stage 37 deg.C for 1 min.
Preferably, the reaction system for competitive allele-specific PCR amplification is 8 μ L: 1.5. mu.L of 80 ng/. mu.L of LDNA, 4. mu.L of 2 XKASP Master mix, 0.14. mu.L of primer mix, and the remainder with ddH2Supplementing and leveling O;
the primer mixture is prepared from 100 mu mol/L TXBH83Fa, TXBH83Fb, TXBH83R and ddH2O is as follows: 12:30:46 in a volume ratio.
The SNP molecular marker related to the Chinese cabbage flower color traits, provided by the invention, has a polymorphism at the 21807238bp position of the A02 chromosome, and specifically has deletion mutation at a G basic group position. Experiments show that the developed SNP molecular marker can obviously distinguish two homozygous genotypes, can identify the heterozygous genotype and has the characteristic of codominant marking, and meanwhile, verification experiments show that the two homozygous genotypes completely accord with the phenotype of the Chinese cabbage flower, when the genotype is the basic group G, the Chinese cabbage flower is expressed as the yellow flower character, and when the genotype is the basic group deletion, the Chinese cabbage flower is expressed as the white flower character. The SNP molecular marker provided by the invention has good universality and accuracy, and can be used for the molecular marker-assisted selection of white petals of Chinese cabbage.
The invention also provides primers for detecting the colors of the Chinese cabbage based on the competitive allele specific PCR technology, which comprise TXBH83Fa, TXBH83Fb and TXBH 83R; the nucleotide sequence of the TXBH83Fa is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the TXBH83Fb is shown as SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the TXBH83R is shown as SEQ ID NO: 4, respectively. The kit comprises a kit body, a kit body and a kit body, wherein TXBH83Fa and TXBH83Fb are two allele specific forward primers, TXBH83Fa is a specific primer of a homozygous yellow flower genotype, TXBH83Fb is a specific primer of a homozygous white flower genotype, and FAM or HEX fluorescent sequence tag sequences. Meanwhile, the primer provided by the invention can accurately identify a large number of materials in a short time, and has the characteristics of high flux and high efficiency.
Drawings
FIG. 1 shows the results of genotyping of KASP primers of the present invention in white cabbage (Y640-288) and yellow cabbage (Y641-87) and F2 isolates;
FIG. 2 is a typing chart of SNP molecular markers in 3F 2 populations, 10 daylily DH lines and 10 albedo DH lines.
Detailed Description
The invention provides an SNP molecular marker related to Chinese cabbage flower color traits, which is positioned in a sequence shown as SEQ ID NO: 1(GTTTCTTGATGTTTTCTCATTTTGACATTACTGATGTTGTTGTTGTTGTTGTCTTTTGAGTAGGGTGTTTGATGTATGGTGCCTTCACATTCCAGTCAGG [ G/- ] ACCGCACTCCTGCTAAAGGTCCCTTCTCTTGCTTTATGTGTTAACCGAGAAGATAGCGTATTGCGGATTAAAGCATTTTCTTTATGTGTTTCAGACTTG), wherein the 101 th base is G/-, where-represents deletion. The invention determines candidate genes of white flower character by a gene positioning method, carries out sequencing and sequence comparison on the candidate genes, and finds that a basic group is deleted at the 21807238bp position of A02 chromosome. The SNP marker A02-21807238 is a deletion of one base G in the white flower material, and when the base G at this position is present, the flower color appears as a yellow flower, and when the base at this position is deleted, the flower color appears as a white flower. Based on the close linkage of the SNP molecular marker and the Chinese cabbage flower color character, the invention provides the application of the SNP molecular marker in the detection of the Chinese cabbage flower color or the breeding of Chinese cabbage plants. The type of the Chinese cabbage is not particularly limited.
The invention provides a primer for detecting Chinese cabbage flower color based on competitive allele specific PCR (KASP), which comprises TXBH83Fa, TXBH83Fb and TXBH 83R. Wherein TXBH83Fa and TXBH83Fb are two allele-specific forward primers and TXBH83R is a specific reverse primer. TXBH83Fa is a specific primer of homozygous yellow flower genotype, and the nucleotide sequence of TXBH83Fa is shown as SEQ ID NO: 2(5' -GAAGGTGACCAAGTTCATGCTCCTTCACATTCCAGTCAGGG-3'); TXBH83Fb is a specific primer of homozygous white flower genotype, and the nucleotide sequence of TXBH83Fb is shown as SEQ ID NO: 3(5' -GAAGGTCGGAGTCAACGGATTCCTTCACATTCCAGTCAGGAC-3'); the nucleotide sequence of the TXBH83R is shown as SEQ ID NO: 4 (5'-CACATAAAGCAAGAGAAGGGACC-3'); wherein the 5' end sequence of the TXBH83Fa or TXBH83Fb is a tag sequence labeled with FAM or HEX fluorescent group. The tag sequences at the 5' ends of the TXBH83Fa and the TXBH83Fb are labeled by different kinds of fluorescent groups, so that the amplified target fragments can read fluorescent signals with different colors. The source of the primer is not particularly limited in the present invention, and a primer known in the art may be used, for example, a primer sequence is artificially synthesized.
And based on the SNP molecular marker capable of effectively distinguishing the color traits of the Chinese cabbage, the application of the primer in the detection of the color of the Chinese cabbage is provided. In view of the requirement of screening white-flower varieties in Chinese cabbage breeding, the primer is applied to Chinese cabbage plant breeding. Meanwhile, the primer is applied to the detection of the Chinese cabbage flower color genotype, and comprises a homozygous genotype and a heterozygous genotype.
The invention provides a kit for detecting the flower color of Chinese cabbage based on competitive allele specificity PCR technology, which comprises the primer. The kit also preferably includes a KASP Master mix. The source of the KASP Master mix is not particularly limited in the present invention, and may be a source of the KASP Master mix known in the art. The method for preparing the kit is not particularly limited, and the method for preparing the kit known in the art can be adopted, for example, each sequence of the synthesized primers is independently packaged, the KASP Master mix is bottled, and each primer and the KASP Master mix are packaged into a packaging box to obtain the kit.
The invention provides application of the kit in detecting the flower color of Chinese cabbage. The invention provides application of the kit in Chinese cabbage plant breeding. Meanwhile, the kit is applied to the detection of the Chinese cabbage flower color genotype, and comprises a homozygous genotype and a heterozygous genotype. The method for detecting the color of the Chinese cabbage is the same as the following method for detecting the color of the Chinese cabbage in high flux.
The invention provides a method for detecting the flower color of Chinese cabbage in high flux, which comprises the following steps:
1) extracting the Chinese cabbage genome DNA of the flower color to be detected;
2) using the genomic DNA in the step 1) as a template, and performing competitive allele specific PCR amplification by using the primer to obtain a KASP amplification product;
3) reading the fluorescence signal of the KASP amplification product by an end point method to obtain a genotyping result;
the genotype fragment obtained by the amplification of the primers TXBH83Fa and TXBH83R is from Chinese cabbage with yellow flower character;
the genotype fragments obtained by the amplification of the primers TXBH83Fb and TXBH83R are from Chinese cabbage with white flower character.
The invention extracts the Chinese cabbage genome DNA of the flower color to be detected. The method for extracting the cabbage genome DNA is not particularly limited, and a DNA extraction kit well known in the field can be adopted. After the Chinese cabbage genome DNA is extracted, preferably, a nucleic acid protein quantifier is adopted to detect the quality and the concentration of the extracted genome DNA, and the qualified DNA is used for subsequent amplification.
After obtaining the genome DNA, the invention takes the genome DNA as a template, and uses the primer to carry out competitive allele specific PCR amplification, so as to obtain KASP amplification products.
In the present invention, the reaction procedure for the competitive allele-specific PCR amplification is preferably: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s, and comprises 10 cycles, and the cycle of the second stage is started, wherein each cycle is reduced by 0.6 ℃; in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; the fourth stage at 37 deg.C for 1 min. The reaction system for competitive allele-specific PCR amplification is preferably 8. mu.L: 1.5 μ L80 ng/. mu.LDNA, 4 μ L2 XKASP Master mix, 0.14 μ L primer mix, remainder with ddH2Supplementing and leveling O; the primer mixture is composed of TXBH83Fa, TXBH83Fb, TXBH83R and ddH with the concentration of 100 mu mol/L2O is as follows: 12:30:46 in a volume ratio. The competitive allele-specific PCR amplification is excellentThe method is carried out in a PCR instrument.
After the KASP amplification product is obtained, the invention reads the fluorescence signal of the KASP amplification product by an end-point method to obtain a genotyping result.
In the present invention, the reading of the end-point fluorescence signal is preferably performed using a Roche fluorescence quantitative PCR Instrument LightCycler480 Instrument II (LC480 II). The SNP typing results were analyzed using LC480 software v1.5.1: the signal point of the homozygous yellow flower material is blue, the 5' end is connected with primer competitive amplification of FAM fluorescent label sequence, and the polymerization is near the X axis; the signal point of the homozygous white flower material is green, the 5' end is connected with primer competitive amplification of the HEX fluorescent label sequence, and the polymerization is near the Y axis; the signal spots of the hybrid yellow material were red and aggregated near the diagonal. The primer can obviously distinguish two homozygous genotypes, can identify the heterozygous genotypes, and the amplified genotypes are completely consistent with the flower color phenotype of the detected Chinese cabbage.
The present invention provides a SNP molecular marker related to the color trait of cabbage, and a detection primer, a detection kit and an application thereof, which will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Test material
Chinese cabbage DH series yellow flower material Y641-87(P1), Y641-87 is obtained by culturing heat-resistant modified Xianpion (purchased from Jinan Shen Germing seed Co., Ltd.) with isolated microspore;
the Chinese cabbage DH line white flower material Y640-288(P2), Y640-288 is obtained by culturing isolated microspore from commercial species Julong Heat-resistant pioneer (purchased from Julong seedling Co., Ltd., Jinan).
2. Color and color character survey
Taking Chinese cabbage DH line yellow flower material Y641-87(P1) and white flower material Y640-288(P2) as parents, respectively carrying out orthogonal and reverse crossing on the two materials to obtain F1 generation seeds, and selfing the F1 material to obtain F2 seeds. Phenotyping was performed on P1, P2, F1 and F2.
The phenotype results show that the petals of 10 yellow flower parents Y641-87 are yellow and 10Petals of the strain white flower parent Y640-288 are white, F1 single strains obtained by positive and negative crossing of 15 strains are yellow petals, the number of the yellow flower single strains and the white flower single strains in the F2 population are 142 and 58 respectively, and the Kafang test (X) is carried out21.71), a separation ratio of 3:1 is met. The white flower character of the Chinese cabbage is controlled by a recessive monogene.
TABLE 1 Chinese cabbage parent and its descendant yellow flower and white flower plant segregation
Example 2
1. Genomic DNA extraction
The modified CTAB method is adopted to extract the genome DNA of each of the two parents, F1 and F2. The method comprises the following specific steps: fresh leaves were taken in a 2.0mL Eppendorf centrifuge tube, 1 bead (5 mm in diameter) was placed in each tube, 1000. mu.L of 2% CTAB extraction buffer was added, and the mixture was disrupted on a tissue disruptor (model: RetschMM400, Germany) for 1min at a frequency of 30 times/sec. Bathing at 65 deg.C for 1.5h, cooling, adding 500 μ L chloroform extract, shaking up and down for 30 times, standing for layering, centrifuging (12000r/min) for 10min, sucking 400 μ L supernatant into new 1.5mL centrifuge tube, adding 400 μ L isopropanol to precipitate DNA, mixing up and down, centrifuging (12000r/min) for 5min, discarding supernatant, adding 750 μ L75% ethanol to wash DNA precipitate, centrifuging (12000r/min) for 2min, discarding supernatant, air drying DNA at room temperature, adding 100 μ L ddH2O dissolving DNA, and placing in a refrigerator at the temperature of 20 ℃ below zero for standby.
Determination of SNP molecular markers
Determining candidate genes of the white flower character by a gene positioning method, sequencing the candidate genes and carrying out sequence comparison on the candidate genes, and finding that a base deletion exists at a 21807238bp position of A02 chromosome, such as SEQ ID NO: 1, a deletion mutation was present in the material of white flowers at 101bp, and this mutation was referred to as SNP marker A02-21807238, and when the base G at this position was present, the flower color appeared as yellow flowers, and when the base at this position was deleted, the flower color appeared as white flowers.
And (3) sequencing results:
GTTTCTTGATGTTTTCTCATTTTGACATTACTGATGTTGTTGTTGTTGTTGTCTTTTGAGTAGGGTGTTTGATGTATGGTGCCTTCACATTCCAGTCAGG[G/-]ACCGCACTCCTGCTAAAGGTCCCTTCTCTTGCTTTATGTGTTAACCGAGAAGATAGCGTATTGCGGATTAAAGCATTTTCTTTATGTGTTTCAGACTTG(SEQ ID NO：1)。
3. development of detection primer TXBH83
A KASP detection primer TXBH83 is designed aiming at the SNP marker A02-21807238 and comprises three primers:
TXBH83Fa:5'-gaaggtgaccaagttcatgctccttcacattccagtcaggg-3'(SEQ ID NO：2)；
TXBH83Fb:5'-gaaggtcggagtcaacggattccttcacattccagtcaggac-3'(SEQ ID NO：3)；
TXBH83R:5'-cacataaagcaagagaagggacc-3'(SEQ ID NO：4)。
wherein, TXBH83Fa and TXBH83Fb are two allele-specific forward primers, TXBH83Fa is a specific primer of a homozygous yellow flower genotype, TXBH83Fb is a specific primer of a homozygous white flower genotype, and FAM and HEX fluorescent sequence tag sequences (underlined parts) are respectively added at the 5' end. TXBH83R is a common reverse primer.
KASP amplification and result analysis
The test was performed on the parental Y641-87, Y640-288 and their F2 progeny individuals using primer TXBH 83. The KASP-PCR reaction was performed on a 96-well PCR instrument with 8. mu.L: 1.5. mu.L of DNA (80 ng. mu.L)-1) 4 μ L of KASP Master mix (2X), 0.14 μ L of primer mix (from 100 μmol. L. concentration)-1TXBH83Fa, TXBH83Fb, TXBH83R and ddH2O is mixed according to the volume ratio of 12:12:30: 46), and the rest is ddH2And (4) supplementing and finishing. The KASP-PCR amplification program was: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s for 10 cycles (from the second cycle, each cycle is reduced by 0.6 ℃); in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; fourth stage 37 deg.C for 1 min.
After the obtained KASP amplification product, the fluorescence signal was read by an end point method using a rowse fluorescent quantitative PCR Instrument LightCycler480 Instrument II (LC480 II). The SNP typing results were analyzed using LC480 software v1.5.1: the signal point of the homozygous yellow flower material is blue, the 5' end is connected with primer competitive amplification of FAM fluorescent label sequence, and the polymerization is near the X axis; the signal point of the homozygous white flower material is green, the 5' end is connected with primer competitive amplification of the HEX fluorescent label sequence, and the polymerization is near the Y axis; the signal spots for the hybrid yellow material were red and aggregated near the diagonal (see FIG. 1). The TXBH83 marker can obviously distinguish two homozygous genotypes, can identify the heterozygous genotype, has the characteristic of codominant marker, and is successfully developed.
Example 3
Kit for detecting cabbage flower color based on competitive allele specificity PCR technology
Primer TXBH83Fa 1 centrifuge tubes;
primer TXBH83Fb 1 centrifuge tubes;
primer TXBH83R 1 centrifuge tubes;
KASP Master mix liquid, available from GmbH scientific and technological Limited, Yong Jia, Beijing.
Example 4
Application of SNP molecular marker detection primer TXBH83 or kit in detection of white flower color characters
1. Extraction of genomic DNA from cabbage leaves by the extraction method described in example 2
The materials are selected from Y509-2, Chiifu, Y66-83, YR16-11 and (Y640-288 XY 509-2) -F2, (Y640-288 XChiifu) -F2, (Y66-83 XYR 16-11) -F2 groups, 10 yellow flower materials: y636-9, Y177-12, Y358-10, Y399-1, Y412-3, Y635-10, Y231-330, YF05-112, SY2004, light 90E 16; 10 white flower materials: 14CR563, 14CR131, Y217-19, Y148-72, Y311-5, Y96-1, Y27-29, Y54-2, Y467-7 and Y81-37.
2. The results of the above material identification were processed using the method of KASP amplification and result analysis described in example 2.
The primers TXBH83 labeled by KASP were verified in 3F 2 populations (Y640-288 XY 509-2-F2, Y640-288 XChiifu-F2, Y66-83 XYR 16-11-F2), 282 individuals in total, and 10 yellow flowers and 10 white flower DH series materials. The results showed that TXBH83 completely matched the genotype and flower color phenotype (FIG. 2), the white flower-appearing material polymerized around the Y-axis, the genotype coincided with Y640-288, and the yellow flower-appearing phenotype polymerized either on the X-axis or around the diagonal. The marker TXBH83 has good universality and accuracy, and can be used for molecular marker-assisted selection of white petals of Chinese cabbage.
The embodiments show that the SNP molecular marker A02-21807238 related to Chinese cabbage flower color traits and the detection primer TXBH83 thereof can accurately and efficiently detect white flower and yellow flower genotypes. The detection method is not influenced by environmental factors, can greatly reduce the workload of field selection, and is beneficial to assisting and accelerating the breeding of the Chinese cabbage.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (9)
1. An SNP molecular marker related to Chinese cabbage flower color traits is characterized in that the SNP molecular marker is positioned in a sequence shown as SEQ ID NO: 1, wherein-represents deletion, and the 101 th base of the gene fragment shown in 1 is G/-.
2. A primer for detecting Chinese cabbage flower color based on competitive allele specificity PCR technology is characterized by comprising TXBH83Fa, TXBH83Fb and TXBH 83R;
the nucleotide sequence of the TXBH83Fa is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the TXBH83Fb is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the TXBH83R is shown as SEQ ID NO: 4 is shown in the specification;
wherein the 5' end sequence of TXBH83Fa or TXBH83Fb is a tag sequence labeled with FAM or HEX fluorescent group.
3. A kit for detecting the flower color of Chinese cabbage based on competitive allele specific PCR technology, which is characterized by comprising the primer of claim 2.
4. The kit of claim 3, wherein the kit further comprises a KASP Master mix.
5. The use of the SNP molecular marker of claim 1, the primer of claim 2 or the kit of claim 3 or 4 for detecting the flower color of Chinese cabbage or detecting the genotype of the flower color of Chinese cabbage.
6. Use of the SNP molecular marker according to claim 1, the primer according to claim 2 or the kit according to claim 3 or 4 in Chinese cabbage plant breeding.
7. A method for detecting the flower color of Chinese cabbage in high flux is characterized by comprising the following steps:
1) extracting the Chinese cabbage genome DNA of the flower color to be detected;
2) performing competitive allele-specific PCR amplification using the genomic DNA of step 1) as a template and the primers of claim 2 to obtain a KASP amplification product;
3) reading the fluorescence signal of the KASP amplification product by an end point method to obtain a genotyping result;
the genotype fragment obtained by the amplification of the primers TXBH83Fa and TXBH83R is from Chinese cabbage with yellow flower character;
the genotype fragments obtained by the amplification of the primers TXBH83Fb and TXBH83R are from Chinese cabbage with white flower character.
8. The method for detecting the flower color of Chinese cabbage according to claim 7, wherein the reaction program of competitive allele-specific PCR amplification is as follows: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s, and comprises 10 cycles, and the cycle of the second stage is started, wherein each cycle is reduced by 0.6 ℃; in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; fourth stage 37 deg.C for 1 min.
9. The method for detecting the flower color of Chinese cabbage according to claim 7 or 8, wherein the reaction system of competitive allele-specific PCR amplification is 8 μ L: 1.5 μ L80 ng/. mu.LDNA, 4 μ L2 XKASP Master mix, 0.14 μ L primer mix, remainder ddH2Supplementing and leveling O;
the primer mixture is prepared from 100 mu mol/L TXBH83Fa, TXBH83Fb, TXBH83R and ddH2And O is mixed according to the volume ratio of 12:12:30: 46.
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