A primer set for identifying flesh color traits of radish fleshy roots and its application

Abstract

The present invention relates to the technical field of radish flesh color identification, and in particular to a primer set, a kit and an application thereof for identifying flesh color traits of radish fleshy roots. The present invention develops molecular markers based on the sequence differences of RsMYB1 and RsF3'H genes between parents, and combines the promoter methylation analysis of RsMYB1 gene to achieve the hybridization of F from Lilimei radish and other types of radish. 2 Compared with single gene RsMYB1 or RsF3'H as molecular markers for radish root color identification, RsMYB1-RsF3'H-RsMYB1 Met The combined analysis significantly improved the accuracy of identifying the flesh color of radish roots in isolated and natural populations.

Description

Technical Field

[0001] The invention relates to the technical field of radish flesh color identification, and in particular to a primer set and a kit for identifying flesh color traits of radish fleshy roots and applications thereof. Background Art

[0002] Radish is an important vegetable crop in the cruciferous family. During the process of evolution, artificial selection and domestication, its edible enlarged fleshy roots have produced rich genetic variation, including root shape, size, color and flavor. Among them, the fleshy root or skin color of Xinlimei radish is red or purple, which is mainly caused by the accumulation of anthocyanins. Anthocyanins are not only an important component of plant color, but also conducive to plant pollination and reproduction, and participate in coping with adverse stress, but also have antioxidant and anti-aging effects, which are beneficial to human health.

[0003] As people's living standards improve, higher requirements are placed on the nutrition and quality of vegetables. Accelerating the cultivation of new varieties of Xinlimei radish with high anthocyanin content and richer colors is an important breeding goal and research hotspot. Among them, the accurate identification of the flesh color of radish fleshy roots is a key technology for breeding high anthocyanin content and rich Xinlimei variety types. Traditional identification of the flesh color of Xinlimei radish fleshy roots requires cutting or drilling parts of the radish, which reduces the survival rate of the plant and makes the breeding material unable to reproduce seeds normally. Therefore, it is urgent to develop relevant molecular markers to control the accumulation of anthocyanins in radish fleshy roots, to assist in the rapid identification of anthocyanin types and contents in fleshy roots.

[0004] Previous studies have shown that the F1 representative of the hybrid between Xinlimei radish and other types of radish (green radish or white radish) has purple skin and purple flesh, and the F2 generation fleshy root flesh color phenotypes include purple, red and white. It is believed that there are two pairs of genes controlling the purple / red traits and the colored / white traits respectively, but their separation ratio is chaotic and does not conform to the strict Mendelian 9:3:4 separation [Masukawa T, Cheon KS, Mizuta D, Kadowaki M, Nakatsuka A, Kobayashi N (2019) Development of mutant RsF3′H allele-based marker for selection of purple and red root in radish (Raphanus sativus L. var. longipinnatus LHBailey). Euphytica, 215, 119.]. The red color of radish fleshy roots is composed of pelargonidin anthocyanins, and the purple color is composed of cyanidin anthocyanins. Flavonoid 3'hydroxylase (F3'H) is the key gene that determines the purple / red trait [Masukawa T, Cheon KS, Mizuta D, Nakatsuka A, Kobayashi N (2017) Insertion of a retrotransposon into a flavonoid 3'hydroxylase homolog confers the redroot character in the radish (Raphanus sativus L. var. longipinnatus L. H. Bailey). The Horticulture Journal 87: 89–96.]. However, the gene that controls the colored / white trait of radish fleshy roots is not clear, and the accuracy of radish fleshy root color identification using RsF3'H as a molecular marker is low due to the confusion of separation ratios, which seriously affects the efficiency and accuracy of using molecular markers to assist in the selection of radish fleshy root color. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a primer set, a kit and an application thereof for identifying the flesh color trait of radish fleshy root. The primer set provided by the present invention can identify the flesh color trait of radish fleshy root, has the advantages of high efficiency, accuracy and few restrictions for the identification of the flesh color trait of radish fleshy root, and provides an effective tool for the identification of the flesh color trait of radish fleshy root and radish breeding.

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

[0007] The invention provides a primer set for identifying flesh color traits of radish fleshy roots, comprising a first primer pair, a second primer pair, a third primer pair and a fourth primer pair;

[0008] The first primer pair includes an upstream primer P1 and a downstream primer P2; the second primer pair includes an upstream primer P3 and a downstream primer P4; the third primer pair includes an upstream primer P5 and a downstream primer P6; the fourth primer pair includes an upstream primer P5 and a downstream primer P7;

[0009] The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the downstream primer P2 is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the upstream primer P3 is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the downstream primer P4 is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of the upstream primer P5 is shown in SEQ ID NO.5;

[0014] The nucleotide sequence of the downstream primer P6 is shown in SEQ ID NO.6;

[0015] The nucleotide sequence of the downstream primer P7 is shown in SEQ ID NO.7.

[0016] Preferably, the primer set further includes a fifth primer pair; the fifth primer pair includes an upstream primer P8 and a downstream primer P9; the nucleotide sequence of the upstream primer P8 is shown in SEQ ID NO.8; the nucleotide sequence of the downstream primer P9 is shown in SEQ ID NO.9.

[0017] The present invention also provides a kit for identifying flesh color traits of radish fleshy roots, and the kit comprises the primer set described in the above technical solution.

[0018] The present invention also provides the use of the primer set described in the above technical solution or the kit described in the above technical solution in identifying the flesh color trait of radish fleshy roots.

[0019] The present invention also provides a method for identifying the flesh color trait of radish fleshy roots, comprising the following steps:

[0020] Using the genomic DNA of the radish to be identified as a template, PCR amplification is performed using the first primer pair, the second primer pair, the third primer pair and the fourth primer pair in the primer set described in the above technical solution respectively;

[0021] Determine the genotype of the radish to be identified according to the PCR amplification results, wherein the determination is to determine the genotype of the radish RsMYB1 gene to be identified according to the amplification results of the first primer pair and the second primer pair, and to determine the genotype of the radish RsF3'H gene to be identified according to the amplification results of the third primer pair and the fourth primer pair;

[0022] The genotype of the RsMYB1 gene is determined as follows: when the first primer pair obtains an amplification product and the second primer pair does not obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / MYB1; when both the first primer pair and the second primer pair obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / myb1; when the first primer pair does not obtain an amplification product and the second primer pair obtains an amplification product, the genotype of the radish RsMYB1 gene to be identified is myb1 / myb1;

[0023] The genotype of the RsF3'H gene is determined as follows: when the third primer pair obtains an amplification product and the fourth primer pair does not obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / F3'H; when both the third primer pair and the fourth primer pair obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / f3'h; when the third primer pair does not obtain an amplification product and the fourth primer pair obtains an amplification product, the genotype of the radish RsF3'H gene to be identified is f3'h / f3'h;

[0024] The flesh color of the fleshy root of the radish to be identified is obtained according to the genotype of the radish to be identified: when the genotype of the RsMYB1 gene of the radish to be identified is myb1 / myb1, the flesh color of the fleshy root of the radish to be identified is white; when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is F3'H / F3'H or F3'H / f3'h, the flesh color of the fleshy root of the radish to be identified is purple; when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is f3'h / f3'h, the flesh color of the fleshy root of the radish to be identified is red.

[0025] Preferably, when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, the method further comprises identifying the methylation degree of the promoter of the RsMYB1 gene: using the genomic DNA of the radish to be identified after bisulfite treatment as a template, and performing PCR amplification using the fifth primer pair in the primer set of claim 2 to obtain an amplified fragment;

[0026] The flesh color of the radish fleshy root to be identified is obtained according to the methylation degree and the genotype result: when the methylation degree of the amplified fragment is greater than 50%, the flesh color of the radish fleshy root to be identified is white.

[0027] Preferably, the reaction systems for PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair are all 10 μL, each including 2 μL of template, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.8 μL of dNTPs, 0.2 μL of Taq enzyme, 1 μL of 10×PCR Buffer and the remainder of ddH2O; the reaction procedures for PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair all include: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0028] Preferably, the source of the genomic DNA of the radish to be identified includes radish tissue.

[0029] The present invention also provides the use of the primer set described in the above technical solution or the kit described in the above technical solution in radish breeding.

[0030] Preferably, the method comprises: using the method described in the above technical solution to identify the flesh color trait of the radish fleshy root to be tested, and selecting purple and / or red as parents for breeding.

[0031] Beneficial effects:

[0032] The invention provides a primer set for identifying flesh color traits of radish fleshy roots, comprising a first primer pair, a second primer pair, a third primer pair and a fourth primer pair; the first primer pair comprises an upstream primer P1 and a downstream primer P2; the second primer pair comprises an upstream primer P3 and a downstream primer P4; the third primer pair comprises an upstream primer P5 and a downstream primer P6; the fourth primer pair comprises an upstream primer P5 and a downstream primer P7; the fifth primer pair comprises an upstream primer P8 and a downstream primer P9; the nucleotide sequence of the upstream primer P1 is shown as SEQ ID NO.1; the nucleotide sequence of the downstream primer P2 is shown as SEQ ID NO.2; the nucleotide sequence of the upstream primer P3 is shown as SEQ ID NO.3; the nucleotide sequence of the downstream primer P4 is shown as SEQ ID NO.4; the nucleotide sequence of the upstream primer P5 is shown as SEQ ID NO.5; the nucleotide sequence of the downstream primer P6 is shown as SEQ ID NO.6; and the nucleotide sequence of the downstream primer P7 is shown as SEQ ID NO.7. The present invention develops molecular markers (first primer pair, second primer pair, third primer pair and fourth primer pair) according to the sequence differences between RsMYB1 and RsF3'H genes in the parents, and combines the promoter methylation analysis of RsMYB1 gene (methylation analysis is performed using the fifth primer pair, recorded as RsMYB1 Met ), which enables accurate identification of the flesh color of the fleshy roots of the F2 generation of hybrids derived from Xinlimei radish and other types of radish (green radish or white radish). Compared with the single gene RsMYB1 or RsF3'H as a molecular marker for radish fleshy root color identification, RsMYB1-RsF3'H-RsMYB1 Met The combined analysis greatly improves the accuracy of identifying the flesh color of radish fleshy roots in isolated populations and natural populations. The primer set provided by the present invention can be used to identify the flesh color trait of radish fleshy roots, and there is no need to cut or drill holes in the radish part, avoiding the problem of low survival rate of the plant and the inability of breeding materials to reproduce seeds normally. The identification of the flesh color trait of radish fleshy roots has the advantages of high efficiency, accuracy and few restrictions, and provides an effective tool for the identification of the flesh color trait of radish fleshy roots and radish breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0034] Figure 1 The fleshy root trait diagram of CMJ01 and R791 parents and some of the constructed F1 and F2 generations;

[0035] Figure 2 The results of QTL-seq analysis of the purple meat pool and red meat pool genomes;

[0036] Figure 3 The results of QTL-seq analysis of the genomes of the color pool (purple meat pool + red meat pool) and the white meat pool;

[0037] Figure 4 The results of association analysis of 9 SNP markers;

[0038] Figure 5 The results of sequence analysis and molecular marker development of RsF3'H and RsMYB1; A is the results of sequence analysis and molecular marker development of RsMYB1; B is the results of sequence analysis and molecular marker development of RsF3'H; C is the electrophoresis diagram after amplification with different primer pairs; the Maker bands in A to C are 2000bp, 1000bp, 750bp, 500bp, 300bp and 100bp from top to bottom respectively. DETAILED DESCRIPTION

[0039] The invention provides a primer set for identifying flesh color traits of radish fleshy roots, comprising a first primer pair, a second primer pair, a third primer pair and a fourth primer pair; the first primer pair comprises an upstream primer P1 and a downstream primer P2; the second primer pair comprises an upstream primer P3 and a downstream primer P4; the third primer pair comprises an upstream primer P5 and a downstream primer P6; the fourth primer pair comprises an upstream primer P5 and a downstream primer P7;

[0040] The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO.1, specifically: 5'-TGATAGCCTGATACCATTGTGC-3';

[0041] The nucleotide sequence of the downstream primer P2 is shown in SEQ ID NO.2, specifically: 5'-TGTAAGTAACGACGCGAGGT-3';

[0042] The nucleotide sequence of the upstream primer P3 is shown in SEQ ID NO.3, specifically: 5'-GACAAGTCCAACTTTGGACTCGGTC-3';

[0043] The nucleotide sequence of the downstream primer P4 is shown in SEQ ID NO.4, specifically: 5'-AAGGCAGGCTTGAGGAAAAG-3';

[0044] The nucleotide sequence of the upstream primer P5 is shown in SEQ ID NO.5, specifically: 5'-ATGACTAATCTCTTCCTCACAATCC-3';

[0045] The nucleotide sequence of the downstream primer P6 is shown in SEQ ID NO.6, specifically: 5'-CTGTCGAACATGTTTAAAATCTTC-3';

[0046] The nucleotide sequence of the downstream primer P7 is shown in SEQ ID NO.7, specifically: 5'-ACCTCCTCTGATGATTTCGAA-3'.

[0047] The present invention develops a molecular marker based on whether there is a 5326bp insertion sequence in the F3'H gene, wherein the electrophoresis band amplified by the third primer pair is about 450bp, which is amplified in purple meat or white meat materials, but not amplified in red meat materials, and is determined to be a dominant genotype F3'H; the electrophoresis band amplified by the fourth primer pair is about 750bp, which is amplified in red meat materials, and in purple meat or white meat materials, there are cases where amplification occurs, and there are cases where amplification does not occur, and is determined to be a recessive genotype f3'h. Therefore, the genotype of the red meat material is f3'h / f3'h, the genotype of the purple meat is F3'H / F3'H or F3'H / f3'h, and the genotype of the white meat is F3'H / F3'H, F3'H / f3'h, or f3'h / f3'h.

[0048] The present invention develops a molecular marker based on whether there is a 7372bp insertion sequence in the RsMYB1 gene, wherein the electrophoresis band amplified by the first primer pair is about 2000bp, which is amplified in the fleshy root purple meat or red meat material, but not amplified in the white meat material, and is determined to be the dominant genotype MYB1; the electrophoresis band amplified by the second primer pair is about 1200bp, which is amplified in the white meat material, and there are cases where amplification occurs in the purple meat or red meat material, and there are also cases where amplification does not occur, and is determined to be the recessive genotype myb1. Therefore, the genotype of the white meat material is myb1 / myb1, and the genotype of the purple meat and red meat is MYB1 / MYB1 or MYB1 / myb1.

[0049] The primer set of the present invention preferably further comprises a fifth primer pair; the fifth primer pair comprises an upstream primer P8 and a downstream primer P9; the nucleotide sequence of the upstream primer P8 is shown in SEQ ID NO.8, specifically: 5'-YYAAAATGYTGTTTTYTTGTAGTGT-3';

[0050] The nucleotide sequence of the downstream primer P9 is shown in SEQ ID NO.9, specifically: 5'-RACCAACRATAAAATATTTRAARTA-3'.

[0051] In the sequences shown in SEQ ID NO.8 and SEQ ID NO.9 of the present invention, "Y" and "R" are degenerate bases, wherein "Y" is C / T and "R" is A / G.

[0052] The present invention proves through experiments that: in very rare cases, when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, due to the methylation of the RsMYB1 promoter, a very small number of colored fleshy root plants will be converted into white fleshy plants. In order to improve the accuracy of using molecular markers to identify the flesh color of radish fleshy roots, the present invention introduces another molecular marker, that is, using the fifth primer pair to analyze the promoter methylation degree of the RsMYB1 gene, which is counted as RsMYB1 Met ; When RsMYB1 Met When it is greater than 50%, it is judged as white meat material.

[0053] The present invention develops molecular markers based on the sequence differences of RsMYB1 and RsF3'H genes between parents, and combines the promoter methylation analysis of RsMYB1 gene to achieve accurate identification of the flesh color of the fleshy roots of the hybrid F2 generation derived from Lilimei radish and other types of radish (green radish or white radish). Compared with the single gene RsMYB1 or RsF3'H as a molecular marker for radish fleshy root color identification, RsMYB1-RsF3'H-RsMYB1 Met The combined analysis significantly improved the accuracy of identifying the flesh color of radish roots in isolated and natural populations.

[0054] The present invention also provides a kit for identifying the flesh color trait of radish fleshy roots, the kit comprising the primer set described in the above technical solution. The kit of the present invention preferably also comprises reagents for PCR amplification; the reagents for PCR amplification preferably comprise: 10×PCRBuffer, dNTPs, Taq enzyme and ddH2O. The present invention has no special requirements on the source and composition of the reagents for PCR amplification, and commercially available products known to those skilled in the art can be used.

[0055] The present invention also provides the use of the primer set described in the above technical solution or the kit described in the above technical solution in identifying the flesh color trait of radish fleshy root. The primer set provided by the present invention can be used to identify the flesh color trait of radish fleshy root, and there is no need to cut or punch holes in the radish part, thereby avoiding the problems of low survival rate of seed plants and inability of breeding materials to reproduce seeds normally. The identification of the flesh color trait of radish fleshy root has the advantages of high efficiency, accuracy and few restrictions, and provides an effective tool for the identification of the flesh color trait of radish fleshy root and radish breeding.

[0056] The present invention also provides a method for identifying the flesh color trait of radish fleshy roots, comprising the following steps:

[0057] Using the genomic DNA of the radish to be identified as a template, PCR amplification is performed using the first primer pair, the second primer pair, the third primer pair and the fourth primer pair in the primer set described in the above technical solution respectively;

[0058] Determine the genotype of the radish to be identified according to the PCR amplification results, wherein the determination is to determine the genotype of the radish RsMYB1 gene to be identified according to the amplification results of the first primer pair and the second primer pair, and to determine the genotype of the radish RsF3'H gene to be identified according to the amplification results of the third primer pair and the fourth primer pair;

[0059] The genotype of the RsMYB1 gene is determined as follows: when the first primer pair obtains an amplification product and the second primer pair does not obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / MYB1; when both the first primer pair and the second primer pair obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / myb1; when the first primer pair does not obtain an amplification product and the second primer pair obtains an amplification product, the genotype of the radish RsMYB1 gene to be identified is myb1 / myb1;

[0060] The genotype of the RsF3'H gene is determined as follows: when the third primer pair obtains an amplification product and the fourth primer pair does not obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / F3'H; when both the third primer pair and the fourth primer pair obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / f3'h; when the third primer pair does not obtain an amplification product and the fourth primer pair obtains an amplification product, the genotype of the radish RsF3'H gene to be identified is f3'h / f3'h;

[0061] The flesh color of the fleshy root of the radish to be identified is obtained according to the genotype of the radish to be identified: when the genotype of the RsMYB1 gene of the radish to be identified is myb1 / myb1, the flesh color of the fleshy root of the radish to be identified is white;

[0062] When the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is F3'H / F3'H or F3'H / f3'h, the flesh color of the fleshy root of the radish to be identified is judged to be purple; when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is f3'h / f3'h, the flesh color of the fleshy root of the radish to be identified is judged to be red.

[0063] In very rare cases, when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, since the methylation of the RsMYB1 promoter will cause a very small number of colored fleshy root plants to be converted into white fleshy plants, in order to improve the accuracy of using molecular markers to identify the flesh color of radish fleshy roots, the method preferably also includes:

[0064] When the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, the methylation degree of the RsMYB1 gene promoter is identified: the genomic DNA of the radish to be identified after bisulfite treatment is used as a template, and PCR amplification is performed using the fifth primer pair to obtain an amplified fragment. When the methylation degree of the amplified fragment is greater than 50%, the flesh color of the fleshy root of the radish to be identified is white.

[0065] In the present invention, the source of the genomic DNA of the radish to be identified preferably includes radish tissue; the radish tissue preferably includes one or more of roots, stems and leaves; more preferably, radish leaves. The present invention selects radish tissues such as roots, stems and leaves to extract genomic DNA, and there is no need to wait until the radish grows fleshy roots and then cut or drill holes in the radish part, which can avoid the problems of low survival rate of the seed plant and the inability of the breeding material to reproduce seeds normally, and the flesh color trait of the fleshy root of the radish can be identified at the plant stage.

[0066] In the present invention, the method for determining the methylation degree of the RsMYB1 gene promoter preferably includes a bisulfite sequencing method; the bisulfite sequencing method preferably includes the following steps:

[0067] Treating the genomic DNA of the radish to be identified with sodium bisulfite;

[0068] The treated genomic DNA was used as a template and PCR amplification was performed using the fifth primer pair. After sequencing the amplified product, the proportion of methylated cytosine (C) in the cytosine site in the sequence was calculated and recorded as RsMYB1. Met .

[0069] In the present invention, the reaction system for PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair is preferably 10 μL, and preferably includes 2 μL of template, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.8 μL of dNTPs, 0.2 μL of Taq enzyme, 1 μL of 10×PCRBuffer and the remainder of ddH2O.

[0070] In the present invention, the reaction procedures for PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, and the fifth primer pair preferably include: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, for 35 cycles; and extension at 72°C for 10 min.

[0071] The present invention also provides the application of the primer group or the kit described in the above technical solution in radish breeding.

[0072] In the present invention, the application preferably includes: identifying the flesh color trait of the to-be-detected radish fleshy root using the method described in the above technical solution, and selecting purple and / or red as parents for breeding.

[0073] The primer group provided by the present invention can quickly and accurately identify the types of anthocyanins in radish fleshy roots, providing an effective tool for breeding purple and / or red materials.

[0074] To further illustrate the present invention, the following describes in detail a primer group, a kit, and their applications for identifying the flesh color trait of radish fleshy roots provided by the present invention with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0075] Example 1

[0076] Development of specific molecular markers for the flesh color trait of radish fleshy roots

[0077] 1. Genetic law of the flesh color trait of radish fleshy roots

[0078] Radish Jingcui No. 2 (CMJ01), showing green skin and red flesh, was purchased from Jingyan Yinong (Beijing) Seed Industry Technology Co., Ltd.; radish "791" (R791), showing green skin and white flesh, was purchased from Zhengzhou Zhengyan Seedling Technology Co., Ltd.

[0079] To clarify the genetic law of the radish fleshy root trait, the present invention used CMJ01 and R791 to construct an F2 population. Without considering the amount of anthocyanin accumulation, the flesh color phenotypes of 225 F2 population individuals could be divided into: purple flesh (PP, 115 plants), red flesh (RR, 35 plants), white flesh (GW, 75 plants) ( Figure 1 ), and the segregation ratio basically conforms to 9:3:4 (χ 2 = 8.531, 0.01 < P = 0.014 < 0.05). The segregation ratio of colored flesh (150 plants) to white flesh (75 plants) in the fleshy root did not conform to 3:1 (χ 2 = 8.333, P = 0.004 < 0.01). The segregation ratio of purple flesh (115 plants) to red flesh (35 plants) in the fleshy root conformed to 3:1 (χ 2=0.222, P=0.637>0.05). These results indicate that the fleshy root color is mainly controlled by two genes, colored root flesh is dominant over white flesh, purple flesh is dominant over red flesh, and the fleshy root color is regulated by other factors, resulting in more white flesh plants in the F2 population.

[0080] 2. Preliminary localization of radish fleshy root color using QTL-seq

[0081] Thirty purple meat, red meat and white meat plants were selected from the above F2 population, and extreme trait pools (purple meat pool, red meat pool and white meat pool) were constructed by mixing equal amounts of DNA. Paired-end (150 bp) sequencing was performed using the Illumina sequencing platform, and read lengths of 56.18 Gb, 37.12 Gb and 49.27 Gb were obtained, respectively. Based on the reference genome "XYB36-2", the average sequencing depths were 89.34×, 63.92× and 81.98×, and the genome coverage was 86.71%, 83.5% and 86.49%, respectively.

[0082] Firstly, QTL-seq analysis was performed using genome resequencing data from the purple meat pool and the red meat pool. A total of 32,607 SNPs were identified. The main QTL controlling the purple flesh trait of radish fleshy root was located in the interval of 20.19-21.65 Mb (p<0.001) on chromosome 7 by calculating the ΔSNP-Index value. The gene was named RsP( Figure 2 ).

[0083] Then, QTL-seq analysis was performed using genome resequencing data from the colored pool (purple meat pool + red meat pool) and the white meat pool. By calculating the ΔSNP-Index value, the major QTL controlling the color (anthocyanin accumulation) trait of radish fleshy roots was located in the intervals of 14.00-14.34Mb, 15.74-16.03Mb and 16.05-16.4Mb (p<0.001) on chromosome 7, and was named gene RsR ( Figure 3 ).

[0084] 3. Fine positioning of genes controlling fleshy root color in radish and prediction of candidate genes

[0085] In order to precisely locate the purple flesh gene RsP in radish fleshy roots, the present invention developed 9 SNP markers in the positioning interval, and the specific information is shown in Table 1. 362 red flesh and purple plants from the F2 population (derived from the hybrid offspring of CMJ01 and R791, 480 plants were sown, and 362 colored plants were selected from them) were screened, and 10 recombinant plants were identified using the RsP site flanking markers.

[0086] Table 1 Primer information of 9 SNP markers

[0087]

[0088]

[0089] Further association analysis located the RsP locus within a 122.8 kb interval between SNP markers R07_21059062 and R07_21181854 ( Figure 4 ). According to the "XYB36-2" reference genome annotation, there are 17 genes in this interval. Gene function annotation shows that gene Rs392880 encodes dihydroflavonol 3'hydroxylase (F3'H, Flavonoid 3'hydroxylase), which is an important structural gene in the anthocyanin synthesis pathway and can catalyze the synthesis of pelargonidin precursor (red) into cyanidin precursor (purple). Therefore, the present invention uses RsF3'H as a key candidate gene for controlling the purple flesh trait of radish fleshy roots.

[0090] In order to obtain the key candidate gene controlling the color (anthocyanin accumulation) trait of radish fleshy root, the present invention performs gene annotation and gene function analysis on the 14.00-14.34Mb, 15.74-16.03Mb and 16.05-16.4Mb regions of chromosome 7 in the initial positioning interval of RsR. There are 140 candidate genes in these intervals, among which Rsa10031919 is annotated as Arabidopsis AtMYB114, which encodes the R2R3-MYB transcription factor family and can regulate the expression of anthocyanin biosynthesis structural genes. Therefore, the present invention uses it as the key candidate gene controlling the color trait (anthocyanin accumulation) of radish fleshy root, and names it RsMYB1.

[0091] 4. Sequence analysis and molecular marker development of genes RsF3'H and RsMYB1 that control radish root color

[0092] The present invention analyzes the sequence of the RsF3'H gene and finds that the full length of RsF3'H in the parent material R791 is 3015 bp, contains an open reading frame of 1536 bp, and encodes a protein with 511 amino acids. Compared with R791, the RsF3'H gene of the parent material CMJ01 contains a 5326 bp insertion sequence in the first exon, and 7 SNPs in the third and fourth exons. Therefore, the present invention develops a molecular marker based on the presence or absence of the 5326 bp insertion sequence ( Figure 5B in the figure). The electrophoretic band amplified by the primer pair P5 / P6 (the nucleotide sequence of primer P5 is shown as SEQ ID NO.5, and the nucleotide sequence of primer P6 is shown as SEQ ID NO.6) is about 450 bp, which is amplified in purple meat or white meat materials, but not in red meat materials, and is determined to be the dominant genotype F3'H; the electrophoretic band amplified by the primer pair P5 / P7 (the nucleotide sequence of primer P5 is shown as SEQ ID NO.5, and the nucleotide sequence of primer P7 is shown as SEQ ID NO.7) is about 750 bp, which is amplified in red meat materials, and may or may not be amplified in purple meat or white meat materials, and is determined to be the recessive genotype f3'h. Therefore, the genotype of red meat materials is f3'h / f3'h, the genotype of purple meat is F3'H / F3'H or F3'H / f3'h, and the genotype of white meat is F3'H / F3'H, F3'H / f3'h, or f3'h / f3'h ( Figure 5 C).

[0093] The present invention analyzes the sequence of the RsMYB1 gene and finds that there is no difference in the gene sequence between the parent materials R791 and CMJ01, both of which are 1276bp, contain 3 exons, and encode 248 amino acids. However, there is a 7372bp CACTA transposon insertion in the promoter region (-154bp) of the RsMYB1 gene of CMJ01. Therefore, the present invention develops a molecular marker ( Figure 5 A in the figure). The electrophoretic band amplified by the primer pair P1 / P2 (the nucleotide sequence of primer P1 is shown in SEQ ID NO.1, and the nucleotide sequence of primer P2 is shown in SEQ ID NO.2) is about 2000bp, which is amplified in the fleshy root purple meat or red meat materials, but not in the white meat materials, and is determined to be the dominant genotype MYB1; the electrophoretic band amplified by the primer pair P3 / P4 (the nucleotide sequence of primer P3 is shown in SEQ ID NO.3, and the nucleotide sequence of primer P4 is shown in SEQ ID NO.4) is about 1200bp, which is amplified in the white meat materials, and may or may not be amplified in the purple meat or red meat materials, and is determined to be the recessive genotype myb1. Therefore, the genotype of the white meat material is myb1 / myb1, and the genotype of the purple meat and red meat is MYB1 / MYB1 or MYB1 / myb1 ( Figure 5 C).

[0094] Then, the present invention used the molecular markers of RsMYB1 and RsF3'H genes to detect 225 plants in the F2 population and found that the consistency between the marker genotype and the plant phenotype reached 99.11%, among which the genotype of two white-fleshed plants was MYB1 / myb1 (plants numbered F2-132 and F2-150 in Table 2). Considering that gene promoter methylation can regulate gene expression, the present invention uses the BS-seq method to analyze the methylation degree of the RsMYB1 gene promoter of these two white meat strains and other 8 strains containing dominant MYB1. The method used is bisulfite sequencing, specifically: 500 ng of genomic DNA of the test material is treated with sodium bisulfite; the treated DNA is amplified by PCR using primer pair P8 / P9 (the nucleotide sequence of primer P8 is shown in SEQ ID NO.8, and the nucleotide sequence of primer P9 is shown in SEQ ID NO.9), and the amplified product is purified and connected to the pMD18-T vector and transformed into the Escherichia coli DH5α strain, 10 positive clones are selected for PCR product sequencing, and finally the proportion of methylated cytosine (C) in the cytosine site in the sequence is calculated according to the sequencing results; the average value of the proportion of all cytosines in the amplified sequence of the P8 / P9 primer pair is taken as the methylation degree of the RsMYB1 gene promoter, recorded as RsMYB1 Met .

[0095] The results showed that the cytosine methylation levels of the RsMYB1 gene promoter in the two white-fleshed plants numbered F2-132 and F2-150 were 56.67% and 62.22%, respectively, while the methylation levels of the RsMYB1 gene promoter in the other eight plants containing dominant MYB1 (purple-fleshed and red-fleshed plants) were between 11.67% and 23.89%, indicating that the methylation of the RsMYB1 gene promoter in the two white-fleshed plants numbered 132 and 150 inhibited the expression of the gene, resulting in the white color of the fleshy roots.

[0096] Therefore, the present invention determines that in the hybrid offspring of CMJ01 (green skin and red flesh) and R791 (green skin and white flesh), due to the methylation of the RsMYB1 promoter, a very small number of colored fleshy root plants will be transformed into white fleshy plants. In order to improve the accuracy of using molecular markers to identify the flesh color of radish fleshy roots, the present invention introduces another molecular marker, that is, the methylation degree analysis of the RsMYB1 gene promoter, that is, RsMYB1 Met The specific judgment criteria are: in very rare cases, when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, when RsMYB1 Met When it is greater than 50%, the flesh color of the radish to be identified is white flesh.

[0097] The amplification systems of P1 / P2, P3 / P4, P5 / P6, P5 / P7 and P8 / P9 are: 2 μl of genomic DNA at a concentration of 50 ng / μl or genomic DNA treated with sodium bisulfite, 1 μl 10×PCR Buffer, 0.8 μl dNTPs, 0.2 μl of upstream primer at a concentration of 10 μM, 0.2 μl of downstream primer at a concentration of 10 μM, 0.2 μl of Taq enzyme at a concentration of 5 U / μl and 5.6 μl ddH2O;

[0098] The amplification procedures for P1 / P2, P3 / P4, P5 / P6, P5 / P7 and P8 / P9 were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0099] The molecular markers of RsMYB1 and RsF3'H genes were used to analyze the methylation degree of the RsMYB1 gene promoter. The genotypes and RsMYB1 expression of 225 radishes with known phenotypes in the F2 population were detected. Met , the results are shown in Table 2.

[0100] Table 2 Identification results of 225 radishes with known phenotypes in the F2 population

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] As shown in Table 2, the RsMYB1-RsF3'H-RsMYB1 provided by the present invention Met The combination of molecular markers can accurately identify the flesh color traits of radish fleshy roots.

[0108] In summary, the primer set (first primer pair, second primer pair, third primer pair, fourth primer pair and fifth primer pair) and identification method provided by the present invention can accurately identify the flesh color traits of radish fleshy roots, and provide an effective tool for the identification of radish fleshy root flesh color traits and radish breeding.

[0109] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer set for identifying fleshy root color traits of radish, characterized in that: comprising a first primer pair, a second primer pair, a third primer pair, a fourth primer pair and a fifth primer pair; The first primer pair includes an upstream primer P1 and a downstream primer P2; the second primer pair includes an upstream primer P3 and a downstream primer P4; the third primer pair includes an upstream primer P5 and a downstream primer P6; the fourth primer pair includes an upstream primer P5 and a downstream primer P7; the fifth primer pair includes an upstream primer P8 and a downstream primer P9; The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer P2 is shown in SEQ ID NO.2; The nucleotide sequence of the upstream primer P3 is shown in SEQ ID NO.3; The nucleotide sequence of the downstream primer P4 is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer P5 is shown in SEQ ID NO.5; The nucleotide sequence of the downstream primer P6 is shown in SEQ ID NO.6; The nucleotide sequence of the downstream primer P7 is shown in SEQ ID NO.7; The nucleotide sequence of the upstream primer P8 is shown in SEQ ID NO.8; The nucleotide sequence of the downstream primer P9 is shown in SEQ ID NO.

9.

2. A kit for identifying the flesh color of radish fleshy roots, characterized in that: The kit comprises the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in identifying the flesh color trait of radish fleshy roots.

4. A method for identifying the flesh color of radish fleshy roots, characterized in that: The following steps are involved: Using the genomic DNA of the radish to be identified as a template, PCR amplification is performed using the first primer pair, the second primer pair, the third primer pair and the fourth primer pair in the primer set of claim 1 respectively; Determine the genotype of the radish to be identified according to the PCR amplification results, wherein the determination is to determine the genotype of the radish RsMYB1 gene to be identified according to the amplification results of the first primer pair and the second primer pair, and to determine the genotype of the radish RsF3'H gene to be identified according to the amplification results of the third primer pair and the fourth primer pair; The genotype of the RsMYB1 gene is determined as follows: when the first primer pair obtains an amplification product and the second primer pair does not obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / MYB1; when both the first primer pair and the second primer pair obtain an amplification product, the genotype of the radish RsMYB1 gene to be identified is MYB1 / myb1; when the first primer pair does not obtain an amplification product and the second primer pair obtains an amplification product, the genotype of the radish RsMYB1 gene to be identified is myb1 / myb1; The genotype of the RsF3'H gene is determined as follows: when the third primer pair obtains an amplification product and the fourth primer pair does not obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / F3'H; when both the third primer pair and the fourth primer pair obtain an amplification product, the genotype of the radish RsF3'H gene to be identified is F3'H / f3'h; when the third primer pair does not obtain an amplification product and the fourth primer pair obtains an amplification product, the genotype of the radish RsF3'H gene to be identified is f3'h / f3'h; The flesh color of the fleshy root of the radish to be identified is obtained according to the genotype of the radish to be identified: when the genotype of the RsMYB1 gene of the radish to be identified is myb1 / myb1, the flesh color of the fleshy root of the radish to be identified is white; when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is F3'H / F3'H or F3'H / f3'h, the flesh color of the fleshy root of the radish to be identified is purple; when the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, and the genotype of the RsF3'H gene is f3'h / f3'h, the flesh color of the fleshy root of the radish to be identified is red; When the genotype of the RsMYB1 gene of the radish to be identified is MYB1 / MYB1 or MYB1 / myb1, the method further comprises identifying the methylation degree of the promoter of the RsMYB1 gene: using the genomic DNA of the radish to be identified after bisulfite treatment as a template, and performing PCR amplification using the fifth primer pair in the primer set of claim 1 to obtain an amplified fragment; The flesh color of the radish fleshy root to be identified is obtained according to the methylation degree and the genotype result: when the methylation degree of the amplified fragment is greater than 50%, the flesh color of the radish fleshy root to be identified is white.

5. The method according to claim 4, characterized in that The reaction system of PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair is 10 μL, including 2 μL of template, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.8 μL of dNTPs, 0.2 μL of Taq enzyme, 1 μL of 10×PCRBuffer and the balance of ddH2O; The reaction procedures for PCR amplification using the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair all include: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 20 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 10 minutes.

6. The method according to claim 4, characterized in that The source of the genomic DNA of the radish to be identified includes radish tissue.

7. Use of the primer set according to claim 1 or the kit according to claim 2 in radish breeding, wherein the use is to identify the flesh color trait of the radish fleshy root to be tested and to select purple and / or red as parents for breeding.

8. The use according to claim 7, characterized in that: include: The method described in any one of claims 4 to 6 is used to identify the flesh color trait of the radish fleshy root to be tested, and purple and / or red are selected as parents for breeding.

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