阅读说明：本技术 一种影响萝卜花青素合成的myb1同源基因及其鉴定方法 (MYB1 homologous gene influencing radish anthocyanin synthesis and identification method thereof ) 是由 陶婧 孙一丁 李石开 汪骞 袁艺 于 2021-09-09 设计创作，主要内容包括：一种影响萝卜花青素合成的MYB1同源基因及其鉴定方法,基于来自云南红萝卜特异种质资源的高代自交系RR1与加工型白萝卜高代自交系WR1杂交产生的F-(2)代遗传群体,构建了相应的遗传图谱,并根据F-(2)代个体的植株(根皮、根肉)花青素含量数据,定位到RsMYB1.3基因是影响萝卜花青素合成的关键基因,无花青素类型萝卜中该基因由于一个InDel插入而提前形成终止密码子,从而导致植株中无花青素合成。依据该基因在双亲本间的序列差异,设计了一组标记,利用该组标记对苗期萝卜植株叶片组织的DNA进行PCR扩增,依照其电泳结果可对RsMYB1.3在无花青素合成萝卜及云南红萝卜中的基因型进行鉴定。(MYB1 homologous gene influencing radish anthocyanin synthesis and identification method thereof, based on F produced by hybridization of high-generation inbred line RR1 from Yunnan red radish specific germplasm resources and processed white radish high-generation inbred line WR1 2 Generating genetic population, constructing corresponding genetic map, and performing genetic mapping according to F 2 The anthocyanin content data of the plant (root bark and root pulp) of the generation individual is positioned to that the RsMYB1.3 gene is a key gene influencing the anthocyanin synthesis of the radish, and the gene in the anthocyanin-free radish forms a stop codon in advance due to one InDel insertion, so that no anthocyanin synthesis exists in the plant. Based on the sequence difference of the gene between two parents, one group of markers is designed and usedAnd performing PCR amplification on DNA of leaf tissues of radish plants in the seedling stage, and identifying the genotypes of RsMYB1.3 in anthocyanin-free synthetic radishes and Yunnan red radishes according to electrophoresis results.)

1. An MYB1 homologous gene RsMYB1.3 affecting anthocyanin synthesis of radishes is characterized in that the gene is an RsMYB1(Loc _108814812) homologous gene and is a key gene affecting anthocyanin synthesis of the radishes, and a stop codon is formed in advance at a 197 th base of the gene in a non-pigment type radishes due to insertion of a 4-bp fragment (5 '-aatt-3'), so that no anthocyanin synthesis exists in plants; no insertion is formed at the 197 th base position of RsMYB1.3 in the Yunnan red radish, so that the expression can be normal, and anthocyanin is generated in a plant.

2. The molecular marker of the gene according to claim 1, wherein the RsMYB1.3-a F/R marker can specifically amplify RsMYB1.3 haplotype gene with 'aatt' base insertion based on the InDel site differential development in the promoter region of the RsMYBl.3 gene, and is suitable for the identification of non-pigment type radish; RsMYB1.3-b F/R can specifically amplify RsMYB1.3 haplotype genes in Yunnan red radish;

the molecular markers are as follows:

RsMYB1.3-a F：5'-GGGAAATGGCACCAAGTTAA-3'；

RsMYB1.3-a R：5'-TCCCAAAAGTTTATGAAGGCGA-3'

RsMYB1.3-b F:5'-ATGCACGTCACTACCCTCATT-3'

RsMYB1.3-b R：5'-TTTAATAAACGACAATCCAACAC-3'。

3. the application of the molecular marker of claim 2 in identifying the color of the fleshy roots of radish.

4. The molecular marker of claim 2, wherein the molecular marker is used for the molecular assisted selection breeding of white-skin radish, white meat radish, green-stem radish, fleshy radish root bark, radish pulp or radish vein.

5. The method for identifying whether a radish plant is colored by using the molecular marker as claimed in claim 2, characterized in that the method takes the genomic DNA of a single plant in a radish variety to be identified or a separation population as a template, and identifies an amplification product by PCR amplification, wherein if an amplification result has a band of only 511bp, the fleshy root and skin and the flesh color of the material to be identified are both white, and the leaf vein of the plant is green; if the 467bp band is amplified from the variety or material to be identified or the 511bp and 467bp bands are simultaneously amplified, the variety or material to be identified is a radish variety or single plant with fleshy root bark, root pulp or leaf vein color.

6. The method of claim 5, wherein the radish material to be identified is from breeding parent material, segregating generation group or radish variety commonly found in the market, and the experiment requires DNA of the material to be tested, which can be extracted from the seed, seedling stage leaf or radish plant in middle and later growth stage of the material to be tested.

Technical Field

The invention relates to a MYB1 homologous gene affecting radish anthocyanin synthesis; in particular to a MYB1 homologous gene influencing the synthesis of anthocyanin of radish and an identification method thereof.

Background

Radish (Raphanus sativusL.) is a brassicaceous radish vegetable, is second to Chinese cabbage in cultivation area throughout the year in China, and plays an important role in agricultural production. The radish planting area is wide, and the planting varieties are various. The Yunnan red radish is a specific germplasm resource rich in anthocyanin (anthocyanidin) represented by a Tonghai black radish and a blue plateau purple radish. Anthocyanins (anthocyanidins) are the largest water-soluble pigments in plants, belong to flavonoid metabolites, and have the effects of inhibiting cell mutation proliferation, resisting inflammation, resisting bacteria, resisting oxidation, reducing blood pressure and the like (He and Giusti, 2010). At present, more than 600 anthocyanidins separated and identified in nature are mainly derived from 6 anthocyanidin aglycones, which are cyanidin aglycone, delphinidin aglycone, pelargonidin aglycone, peoniflorin aglycone, petunidin aglycone and malvidin aglycone (Kong et al, 2003; Daselan & Hongyan, 2016). Anthocyanins in radish are mainly derived from purple cyanidin aglycone and red pelargonidin glycoside (Kato et al, 2013). The red radish pigment extracted from Yunnan red radish has bright red color and good stability, and is widely used in food, medicine, cosmetics and other industries. Since the 90 s of the 20 th century, Yunnan province has vigorously developed the radish red pigment processing industry in Tonghai county, Lanping county and Dongchua city, and the products have been certified for origin marking. By 2019, 100 tons of radish red pigment are produced in Yunnan province, the direct production value is over one hundred million yuan, more than 90 percent of primary processed products are sold to the places of America, Japan, Europe, Korea and the like, and the international market share is more than 80 percent. Because the planting and processing technology is simple and the benefit is stable, the carrot has been developed into a characteristic and advantageous industry for promoting the continuous income increase of farmers in mountainous areas in the Yunnan vegetable industry. However, the variety and germplasm of the red radish used in production are mixed, the root shape difference is large, the pigment content is uneven, and the processing characteristics are uneven, so that the difficulty of the extraction process of the red radish pigment is increased, the improvement of the product quality is hindered, and the further development of the red radish pigment industry is restricted. Therefore, key regulation genes for synthesizing anthocyanin of the fleshy roots of the Yunnan red radish are excavated, the regulation mechanism of the regulation genes is analyzed, and corresponding molecular markers are developed on the basis of the regulation genes for auxiliary breeding, so that theoretical basis and technical support are provided for genetic improvement of the Yunnan red radish.

At present, researchers at home and abroad also carry out related research around the important commodity character of radish meat quality and color, and the genetic diversity and karyotype analysis results show that the genetic relationship among radish varieties with different meat colors is relatively close (Xijiang et al, 2011; Fang et al, 2012); the pigment appears after the red fleshy radish germinates for 2-3 days, which shows that the synthesis of anthocyanin is started in the early germination stage and the distribution pattern is fixed (Lusheng et al, 2006). Transcriptome analysis shows that the expression levels of RsDFR, RsANS, RsUFGT, RsF3H, RsCHS3 and RsF 3' H1 in red fleshy radish are higher than those of other types of materials, wherein the expression of RsUFGT is a key control point of anthocyanin spatiotemporal accumulation (Park et al, 2011; Muleke et al, 2017; Sun et al, 2018). Exogenous methyl jasmonate, gibberellin and UV-A can induce white fleshy radishes to generate and accumulate anthocyanin (Su et Al 2014; Al-Dhabi et Al 2015), which indicates that different fleshy radishes have related structural genes for synthesizing anthocyanin and only have different mechanisms for regulating and controlling the expression of anthocyanin. Recent research shows that the transcription factor RsMYB1/90 can directly regulate the expression of anthocyanin synthesis structural genes, is a key gene (Lim et al, 2016, 2017; Luo et al, 2019) for determining radish skin color, and RsMYB1 can directly regulate the expression of bHLH transcription factor in transgenic arabidopsis; the bHLH transcription factor RsTT8 can form a MYB-bHLH-WDR (MBW) complex with RsMYB1/90, and jointly regulate radish pigment synthesis (Yi et al.2018). Lai et al (2019) identified two RsMYB1/90 homologous genes by homologous cloning, wherein MYB1a in the second linkage group is a key gene for production of Fuling carmine radish anthocyanin; epigenetic studies found that methylation by insertion of a CACTA transposon in the promoter region of the MYB1/90 gene located in linkage group 7 resulted in increased expression of MYB1/90, which is closely related to the red meat trait of Yukamei radish (Wang et al, 2020). The radish root flesh color trait is complicated in heredity, and the research result cannot completely disclose an anthocyanin synthesis regulation mechanism. In addition, the Yunnan red radish is mostly distributed on low latitude plateaus with the altitude of more than 1800m, is in the ecological environment with high ultraviolet radiation and large day-night temperature difference for a long time, and has high anthocyanin content and great differences in properties such as leaf shape, root shape, skin color and the like compared with the northern Xinlimei radish which is researched more at present.

In order to clarify the molecular mechanism of anthocyanin specific accumulation in the fleshy root of Yunnan red radish, the inventor of the invention uses the high-generation inbred line RR1(P1) bred from the local material of Yunnan red radish to hybridize with the high-generation inbred line WR1(P2) of the processed radish (white-skin white meat) to create BC₁P₁、BC₁P₂、F₁、F₂Genetic population, and for two parent materials and 200F₂The generation material is subjected to re-sequencing and SLAF simplified genome sequencing respectively, and F is utilized₂The genetic population constructs a high-density genetic map and researches the QTL positioning of anthocyanin trait association of the fleshy root.

Phenotypic observations revealed that F does not take into account the site and extent of coloration between individuals₂In the genetic population, the ratio of the number of individuals with the color trait (Pi) to the number of individuals with the colorless trait (non-trait, NP) corresponds to a Mendelian segregation ratio of 3:1, while at BC₁P₁All individuals in the population have color, whereas at BC₁P₂The ratio of the number of Pi individuals to the number of NP individuals in the population was 1:1, indicating that in the constructed genetic population, radish fleshy root pigment synthesis was controlled by a major monogene.

Binding F₂The individual's anthocyanin content and genetic map constructed, the LOD threshold corresponding to 0.99 confidence level, detected on LG07And (4) QTL. Mapping the locus to a physical map of the radish genome by using published radish genome data, and preliminarily deducing that the candidate gene for controlling the accumulation of the anthocyanin of the radish is RsMYB1.3 by functional annotation of candidate genes of nearby loci. Semi-quantitative PCR experiments showed that rsmyb1.3 is expressed in RR1, but not in WR 1; the structures of RsMYB1.3-RR1 and RsMYB1.3-WR1 were analyzed, and it was found that there was a 4bp base insertion in the first exon of RsMYB1.3-WR1, which resulted in the premature formation of a stop codon and loss of function of RsMYB1.3-WR 1. Aiming at the insertion and the structural characteristics of RsMYB1.3-RR1 and RsMYB1.3-WR1, two pairs of primers RsMYB1.3-a F/R and RsMYB1.3-b F/R are designed, wherein RsMYB1.3-a F/R can be specifically amplified from RsMYB1.3-WR1 to obtain 511bp fragments, and RsMYB1.3-b F/R can be specifically amplified from RsMYB1.3-RR1 to obtain 467bp fragments. Using the two primer pairs F₁The plant material is amplified, and F is found₁511bp and 467bp bands can be obtained by amplification in generation materials, which shows that RsMYB1.3 is in F₁The generation material is in a hybrid state. At F₂In the generation group, only 511bp fragments can be amplified in NP character individuals, two forms of 467bp and 511bp/467bp can be obtained in Pi character individuals, therefore, the genotype of RsMYB1.3 in the genetic material can be judged by using the amplification results of the two pairs of primers, and the amplified fragments of the two pairs of primers can be used as functional markers to screen the genetic material with anthocyanin synthesis function.

In the breeding and variety improvement process work aiming at the aspects of the quality, the yield and the like of Yunnan red radish, breeding materials need to be hybridized or backcrossed to create a large amount of breeding intermediate materials, the traditional identification method for the color of the fleshy root of the materials is to cut and observe the color of the fleshy root after the radish is pulled out of the ground, the method wastes time and labor, the fleshy root is damaged, the survival rate after transplantation is low, and the loss of the excellent breeding intermediate materials is caused.

The existing identification scheme for red flesh color and purple skin color of radish fleshy roots by using molecular markers comprises the following steps:

molecular marker for identifying red flesh color of radish fleshy root "

Specific molecular marker for identifying radish fleshy root purple peel character "

In the breeding and variety improvement process aiming at the aspects of the quality, the yield and the like of Yunnan red radish, breeding materials need to be hybridized or backcrossed, a large amount of breeding intermediate materials are created, the traditional identification method of the color of fleshy roots of the materials is to pull the radish out of the ground, cut and observe the color of the fleshy roots, and then transplant the radish into soil to wait for pollination and seed collection. The method is time-consuming and labor-consuming, and can cause damage to fleshy roots, low survival rate after transplantation and loss of good breeding intermediate materials. According to the invention, radish germplasm resources, breeding materials and separated progeny plants are identified early by the marker, so that the planting and identification scale of the plants of the materials to be identified can be reduced, the investment of manpower, material resources and financial resources in the identification and cultivation management processes is greatly reduced, the problems of difficult storage and low survival rate of damaged seed roots caused by cutting and observing the fleshy roots by the traditional root flesh color identification method are solved, and the selection efficiency is improved; in addition, the marker can be used for molecular marker-assisted breeding of radish chromogenic traits, and genetic improvement efficiency of complex traits is improved.

The existing scheme for identifying radish red fleshy roots by molecular markers has the following defects: (1) the located functional gene is different from the gene of the research, and the genotype difference exists in different radish germplasms due to the genetic mechanism generated by the anthocyanin of the radish, so that the insertion of the RsMYB1.3 inactivated 4-bp base is discovered for the first time. (2) The detection method is different, the method specifically detects the insertion site of the 4-bp base causing the inactivation of RsMYB1.3, and the current method detects the sizes of the MYB gene introns at different chromosome positions.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to perform PCR amplification on DNA of a leaf tissue of a radish plant at a seedling stage by using a design marker, and can identify the genotype of RsMYB1.3 in anthocyanin-free synthetic radish and Yunnan red radish according to an electrophoresis result. The marker obtained by the invention can be used for carrying out early identification on radish germplasm resources, breeding materials and separated progeny plants, so that the planting and identification scales of the plants of the materials to be identified are reduced, the workload and the investment of material resources and financial resources in the identification process are greatly reduced, and the selection efficiency is improved; meanwhile, the method can also solve the problems of difficult seed root storage and low survival rate caused by cutting and observing the fleshy root by the traditional fleshy root color identification method.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an MYB1 homologous gene RsMYB1.3 affecting anthocyanin synthesis of radishes is characterized in that the gene is an RsMYB1(Loc _108814812) homologous gene and is a key gene affecting anthocyanin synthesis of the radishes, and a stop codon is formed in advance at a 197 th base of the gene in a non-pigment type radishes due to insertion of a 4-bp fragment (5 '-aatt-3'), so that no anthocyanin synthesis exists in plants; no insertion is formed at the 197 th base position of RsMYB1.3 in the Yunnan red radish, so that the expression can be normal, and anthocyanin is generated in a plant.

The molecular marker based on the gene of claim 1, which is characterized in that based on the InDel locus difference development in the promoter region of the RsMYBl.3 gene, the RsMYB1.3-a F/R marker can specifically amplify the RsMYB1.3 haplotype gene with 'aatt' base insertion, and is suitable for the identification of the pigment-free radish; RsMYB1.3-b F/R can specifically amplify RsMYB1.3 haplotype genes in Yunnan red radish.

The molecular markers are as follows:

RsMYB1.3-a F：5'-GGGAAATGGCACCAAGTTAA-3'；

RsMYB1.3-a R：5'-TCCCAAAAGTTTATGAAGGCGA-3'

RsMYB1.3-b F:5'-ATGCACGTCACTACCCTCATT-3'

RsMYB1.3-b R：5'-TTTAATAAACGACAATCCAACAC-3'

further, the molecular marker is applied to the identification of the color of the fleshy roots of radish.

Further, the method for identifying whether the radish plant is colored based on the molecular marker is characterized in that genomic DNA of a single plant in a variety or a segregation population of the radish to be identified is used as a template, an amplification product is identified through PCR amplification, and if an amplification result is provided and only 511bp bands exist, the fleshy root bark and flesh color of the material to be identified are white, and the vein of the plant is green; if the 467bp band is amplified from the variety or material to be identified or the 511bp and 467bp bands are simultaneously amplified, the variety or material to be identified is a radish variety or single plant with fleshy root bark, root pulp or leaf vein color.

Furthermore, the radish material to be identified in the method is from breeding parent material, segregating generation group or common radish variety in the market, DNA of the material to be detected is needed in the experiment, and the DNA can be extracted from seeds, seedling stage leaves or radish plants in the middle and later growth stages of the material to be detected.

Furthermore, the molecular marker is applied to molecular assisted selective breeding of white-skin radish, white meat radish, green stem or fleshy radish roots, meats or veins.

Compared with the prior art, the invention has the beneficial effects that:

the DNA of the leaf tissue of the radish plant at the seedling stage is subjected to PCR amplification by using a design marker, and the genotype of RsMYB1.3 in the anthocyanin-free synthetic radish and Yunnan red radish can be identified according to the electrophoresis result. The marker obtained by the invention can be used for carrying out early identification on radish germplasm resources, breeding materials and separated progeny plants, so that the planting and identification scales of the plants of the materials to be identified are reduced, the workload and the investment of material resources and financial resources in the identification process are greatly reduced, and the selection efficiency is improved; meanwhile, the method can also solve the problems of difficult seed root storage and low survival rate caused by cutting and observing the fleshy root by the traditional fleshy root color identification method.

The existing scheme for identifying radish red fleshy roots by molecular markers has the following defects: the located functional genes are different from the genes obtained by the research, and because the genetic mechanism for generating the anthocyanin of the radish has genotype difference in different radish germplasms, the existing markers cannot be utilized in the genetic breeding of the specific resource of the Yunnan red radish.

Drawings

FIG. 1.A. parent inbred lines and F₂Phenotype of the individual. (a) The parent strain 'YAAS-RR 1'. (b) The parent strain ` YAAS-WR1 `. (d-l) F of different colors₂Phenotype of the individual. B.F₂For individual pigmentary skin characteristicsA frequency distribution. C.F₂Frequency distribution of individual meat pigment traits.

FIG. 2 QTL location related to radish coloration. The abscissa indicates the order of the markers in the linkage group. The upper and lower ordinates represent the LOD values, the middle ordinate represents the phenotypic contribution rate. The curves of the upper and lower panels represent the genetic coordinates and LOD score (top) of the QTL detected. The blue line is the LOD value corresponding to the mark; the red line is the corresponding phenotype contribution rate of the marker; the gray line is the threshold line.

FIG. 3. Gene expression profiles among parent materials based on semi-quantitative PCR analysis for RsMYB1.3.

FIG. 4 alignment of radish and carrot RsMYB1.3 sequences. "-" indicates the same base as the allele LOC202036 in the reference genome. "." indicates the missing base. The premature stop codon is indicated in red box.

FIG. 5 development of primer PCR marker F₁、F₂The result of the individual. (a represents a primer RsMYB 1.3-aF/R; b represents a primer RsMYB 1.3-bF/R; the numbers 3, 4, 17, 106, 184 and 196 respectively represent fleshy F with red root bark, root pulp or veins₂Carrying out single plant cultivation; the numbers of 6, 9, 25, 102, 163 and 175 represent fleshy root bark, root pulp or purple-vein F₂Carrying out single plant cultivation; f of white bark, white meat and green stem with numbers of 5, 36, 47, 91 and 93 respectively₂Single plant)

Detailed Description

The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:

as shown in the figures 1-5 of the drawings,

example 1: MYB1 homologous gene influencing radish anthocyanin synthesis and molecular marker development thereof

To elucidate the molecular mechanism of anthocyanin specific accumulation in the fleshy root of Yunnan red radish, the inventor utilized the high-generation inbred line RR1 (P) bred from the local material of Yunnan red radish₁) Mixing with processed radish (white skin and white meat) high-generation inbred line WR1 (P)₂) Hybridization is carried out to create BC₁P₁、BC₁P₂、F₁、F₂Genetic populationAnd for two parent materials and 200F₂The generation material is subjected to re-sequencing and SLAF simplified genome sequencing respectively, and F is utilized₂The genetic population constructs a high-density genetic map and researches the QTL positioning of anthocyanin trait association of the fleshy root.

Phenotypic observations revealed that F does not take into account the site and extent of coloration between individuals₂In the genetic population, the ratio of the number of individuals with the color trait (Pi) to the number of individuals with the colorless trait (non-trait, NP) corresponds to a Mendelian segregation ratio of 3:1 (Table 1), while at BC₁P₁All individuals in the population have color, whereas at BC₁P₂The ratio of the number of Pi individuals to the number of NP individuals in the population was 1:1 (Table 1), indicating that radish sarcomeric root pigment synthesis is controlled by a major single gene in the constructed genetic population.

Binding F₂The individual anthocyanin levels and the constructed genetic profile, LOD threshold, corresponded to a 0.99 confidence level, with a QTL detected on LG07 (fig. 2). Mapping the locus to a physical map of the radish genome by using published radish genome data, and preliminarily deducing that the candidate gene for controlling the accumulation of the anthocyanin of the radish is RsMYB1.3 by functional annotation of candidate genes of nearby loci. Semi-quantitative PCR experiments showed that rsmyb1.3 is expressed in RR1 but not in WR1 (fig. 3); analysis of the structures of RsMYB1.3-RR1 and RsMYB1.3-WR1 revealed a 4bp base insertion in the first exon of RsMYB1.3-WR1 (FIG. 4), which resulted in the premature formation of a stop codon and loss of function of RsMYB1.3-WR 1. Aiming at the insertion and the structural characteristics of RsMYB1.3-RR1 and RsMYB1.3-WR1, two pairs of primers RsMYB1.3-a F/R and RsMYB1.3-b F/R are designed, wherein RsMYB1.3-aF/R can be specifically amplified from RsMYB1.3-WR1 to obtain 511bp fragments, and RsMYB1.3-bF/R can be specifically amplified from RsMYB1.3-RR1 to obtain 467bp fragments.

TABLE 1.6 genetic groups radish pigment phenotypic isolation characterization

Example 2: verification of MYB1 homologous gene molecular marker affecting radish anthocyanin synthesis in progeny population

Using the two primer pairs F₁The plant material is amplified, and F is found₁The generation material can be amplified to obtain 511bp and 467bp bands (FIG. 5), which shows that RsMYB1.3 is in F₁The generation material is in a hybrid state. At F₂Only 511bp fragments could be amplified in the individuals with NP trait in the generation population, while 467bp and 511bp/467bp two forms could be obtained in the individuals with Pi trait (FIG. 5), indicating F₂The RsMYB1.3 locus in the generation group material has both heterozygous individuals and homozygous individuals. Therefore, the two pairs of primers are used for amplification, the genotype of RsMYB1.3 in the genetic material can be judged according to the result, and the amplified fragments of the two pairs of primers can be used as functional markers for screening the genetic material with the anthocyanin synthesis function.

The RsMYB1.3 haplotype gene (RsMYB1.3-WR1) of the radish without the anthocyanin accumulation character loses function due to the insertion of 4-bp base, so that the radish plant cannot synthesize anthocyanin. RsMYB1.3-WR1 is obtained by first identification, is widely distributed in natural radish groups, and has great utilization value for breeding work.

Providing a group of PCR primer sequences for identifying the radish pigment synthesis determining gene RsMYB1.3 genotype:

RsMYB1.3-a F：5'-GGGAAATGGCACCAAGTTAA-3'；

RsMYB1.3-a R：5'-TCCCAAAAGTTTATGAAGGCGA-3'

RsMYB1.3-b F:5'-ATGCACGTCACTACCCTCATT-3'

RsMYB1.3-b R：5'-TTTAATAAACGACAATCCAACAC-3'

FIG. 1.A. parent inbred lines and F₂Phenotype of the individual. (a) The parent strain 'YAAS-RR 1'. (b) The parent strain ` YAAS-WR1 `. (d-l) F of different colors₂Phenotype of the individual. B.F₂Frequency distribution of individual pigmented skin traits. C.F₂Individual pigmentFrequency distribution of meat traits.

FIG. 2 QTL location related to radish coloration. The abscissa indicates the order of the markers in the linkage group. The upper and lower ordinates represent the LOD values, the middle ordinate represents the phenotypic contribution rate. The curves of the upper and lower panels represent the genetic coordinates and LOD score (top) of the QTL detected. The blue line is the LOD value corresponding to the mark; the red line is the corresponding phenotype contribution rate of the marker; the gray line is the threshold line.

FIG. 3. Gene expression profiles among parent materials based on semi-quantitative PCR analysis for RsMYB1.3.

FIG. 4 alignment of radish and carrot RsMYB1.3 sequences. "-" indicates the same base as the allele LOC202036 in the reference genome. "." indicates the missing base compared to the other alleles. The premature stop codon is indicated in red box.

FIG. 5 development of primer PCR marker F₁、F₂The result of the individual. (a represents a primer RsMYB1.3-a F/R; b represents a primer RsMYB1.3-b F/R; the numbers 3, 4, 17, 106, 184 and 196 respectively represent fleshy root bark, root pulp or F with red veins₂Carrying out single plant cultivation; the numbers of 6, 9, 25, 102, 163 and 175 represent fleshy root bark, root pulp or purple-vein F₂Carrying out single plant cultivation; f of white bark, white meat and green stem with numbers of 5, 36, 47, 91 and 93 respectively₂Single plant)

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.