Molecular marker closely linked with radish green meat character and application thereof

Abstract

The invention provides a molecular marker closely linked with radish green meat traits and application thereof, and belongs to the technical field of nucleic acid-containing determination methods, the molecular marker is an Indel molecular marker, and the nucleotide sequence of the Indel molecular marker is as shown in SEQ ID NO.4 in a sequence table; a primer for amplifying the molecular marker is used for detecting an Indel site; the polymorphism of the Indel site is a deletion or insertion sequence, and the nucleotide sequence of the insertion sequence is shown as SEQ ID NO.1 in a sequence table. A primer for amplifying the Indel molecular marker is used for identification in 53 parts of radish materials, and the coincidence rate of the identification result and the phenotype of the radish reaches 94.3%.

Description

Technical Field

[0001] The invention relates to a molecular marker closely linked to a green flesh trait of radish and application thereof, belonging to the technical field of determination methods involving nucleic acids. Background Art

[0002] Radish is a popular vegetable with a long history of cultivation in my country. The fleshy root comes in white, green, red, etc. Different flesh colors of radishes have different tastes, uses, and nutritional values. For example, white radish tastes slightly sweet and is suitable for stewing; green radish is crisp, sweet, and juicy, suitable for eating raw; red radish is rich in anthocyanins and has a higher nutritional value.

[0003] The main method for identifying the flesh color of radish is to cut the fleshy roots of radish during the mature stage and then conduct phenotypic identification. This is a destructive detection method and requires a lot of time and manpower. It is inefficient, and the identification results are easily affected by personal experience and are highly subjective. However, the use of molecular marker detection can screen the flesh color of radish at an early stage, shorten the breeding cycle, reduce costs, and the results are more stable and reliable. More importantly, it will not damage the radish, can maintain the integrity of the radish, and is more conducive to the survival of the plant and the screening of commercial radish.

[0004] There are many types of molecular markers, and Indel (Insertion-Deletion) molecular markers are one of the common markers. Indel molecular markers, or insertion-deletion markers, refer to polymorphisms caused by the insertion or deletion of DNA fragments at the genomic level. It is a molecular marker based on PCR technology, widely distributed in the genome, and has the advantages of rich polymorphism, high stability, and simple operation.

[0005] CN110229923A discloses a molecular marker for identifying the red flesh color of radish fleshy roots, which is used to distinguish the red flesh and white flesh traits of radish; CN116875737A discloses a primer set for identifying the flesh color trait of radish fleshy roots and its application, which is used to identify the red flesh, purple flesh and white flesh traits of radish.

[0006] Among the molecular markers for identifying fleshy roots of radish in the prior art, there is no report on identifying the green flesh trait of radish. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a molecular marker closely linked to the green flesh trait of radish and its application, to achieve the following invention objectives: the green flesh trait of radish can be detected with high accuracy of the detection result.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: A molecular marker tightly linked to a green flesh trait of radish, the molecular marker being an Indel molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.4 in the sequence table; a primer for amplifying the molecular marker is used to detect an Indel site; the polymorphism of the Indel site is a deletion or insertion sequence, the nucleotide sequence of which is shown as SEQ ID NO.1 in the sequence table.

[0009] The Indel site is located at positions 45402470-45402485 of the full-length sequence of chromosome 6 of the radish reference genome.

[0010] The primers for amplifying the molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.5 in the sequence list; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6 in the sequence list.

[0011] The primers for amplifying the molecular markers are used in identifying radish flesh traits.

[0012] The radish flesh is green or white.

[0013] The application comprises the following steps: (1) Extracting genomic DNA from the radish leaves to be tested; (2) Using genomic DNA from radish leaves as a template, PCR amplification was performed using primers for amplifying molecular markers, and the PCR amplification products were detected; (3) If an amplification product with a length of 969 bp is amplified, and the nucleotide sequence of the amplification product is as shown in SEQ ID NO.4 in the sequence listing, the radish flesh trait to be detected is white flesh; if no amplification product is amplified, the radish flesh trait to be detected is green flesh.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention screens an Indel site that is closely linked to the green flesh trait of radish, designs an Indel molecular marker and a primer for amplifying the molecular marker based on the site, and uses the primer to perform amplification in the radish DNA to be detected, and can identify the green flesh trait of the radish based on the situation of the amplified product with high accuracy.

[0015] The primers for amplifying the Indel molecular marker of the present invention were used to conduct identification in 53 radish materials, and the identification results were consistent with the phenotype of radish at a rate of 94.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the distribution map of Indel sites on the 9 chromosomes of the radish reference genome; Figure 2 This is the electrophoresis diagram of the amplification of Indel-1 marker in radishes No. 1-6; Among them, No. 1-3 are green-fleshed radishes, and No. 4-6 are white-fleshed radishes; Figure 3 This is the electrophoresis diagram of the amplification of Indel-1 marker in radish No. 7-11; Among them, No. 7-10 are green-fleshed radishes, and No. 11 is white-fleshed radish; Figure 4 This is the electrophoresis diagram of the amplification of Indel-1 marker in radish No. 12-26; Among them, radishes with numbers 12-18 are green flesh, and radishes with numbers 19-26 are white flesh; Figure 5 This is the electrophoresis diagram of the amplification of Indel-1 marker in radish No. 27-39; Among them, radishes with numbers 27-30 are green flesh, and radishes with numbers 31-39 are white flesh; Figure 6 This is the electrophoresis diagram of the amplification of Indel-1 marker in radish No. 40-53; Among them, No. 40-50 are green-fleshed radishes, and No. 51-53 are white-fleshed radishes. DETAILED DESCRIPTION

[0017] Example 1 Development of Indel Molecular Markers 1. Indel Identification The 290 radish materials used in the present invention are composed of local radish varieties, bred varieties and advanced inbred lines, etc. The material numbers are shown in Tables 1 to 4, including 187 white flesh radishes, 53 green flesh radishes, 12 red flesh radishes and 38 purple flesh radishes. The above materials are collected and preserved by the National Germplasm Resource Bank of the Chinese Academy of Agricultural Sciences, Weifang University and Weifang Academy of Agricultural Sciences.

[0018] Table 1 Numbering information of radish materials from 1st to 76th

[0019] Table 2 Number information of radish materials from 77 to 152

[0020] Table 3 Number information of radish materials from 153 to 228

[0021] Table 4 Number information of radish materials No. 229-290

[0022] The above 290 materials were resequenced using the Illumina Novaseq 6000 sequencing platform, and a total of 1597GB of high-quality base data were obtained, with an average sequencing depth of 12.96x and an average Q30 of 91.54%. The sequence reads obtained from the previous sequencing were aligned to the radish reference genome Rs1.0 (http: / / radish-genome.org / ) using the BWA software, and Indel identification was performed using the Gatk4 program.

[0023] Indels were filtered and quality controlled using VCF tools software, and Indel sites that met the following criteria were retained: ① the average sequencing depth of all sites was greater than 4×; ② the maximum deletion rate was 5%; ③ the completeness was greater than 80%; ④ only dialleles were retained. In the end, a total of 308,324 Indel sites distributed on the 9 chromosomes of the radish reference genome were obtained ( Figure 1 ).

[0024] 2. GWAS analysis of green flesh traits of radish Tassel 5.0 software was used to conduct genome-wide association analysis using the GLM+PCA model combined with the flesh color trait of radish fleshy roots, and the P-value threshold was set at 1×10 -7 , and finally obtained 1697 Indel sites significantly associated with green meat traits.

[0025] 3. Screening of Indel sites The above 1697 Indel sites were analyzed one by one, and Indel sites that met the following criteria were selected: ① Indel fragment length ≥ 6bp; ② The consistency rate between Indel genotype and radish green / white flesh color phenotype ≥ 90%.

[0026] Finally, 8 Indel sites were screened (see Table 5).

[0027] Table 5 Eight Indel sites significantly associated with green flesh traits of radish

[0028] According to the physical position of the Indel site, 1000bp DNA sequences upstream and downstream of the reference genome were extracted, and primers were designed. Then, using the genomic DNA of green-fleshed and white-fleshed radishes as templates, PCR amplification and product detection were performed using primers of the above-mentioned Indel sites. The results showed that only the Indel-1 site could well distinguish the green flesh and white flesh traits of radish; the other Indel-2 to Indel-8 could not well distinguish the green flesh and white flesh traits of radish.

[0029] Finally, the Indel-1 site was selected, which is an Indel site closely linked to the green flesh trait of radish.

[0030] 4. Development of molecular markers and primers The Indel-1 site is located at positions 45402470-45402485 of the full-length sequence of chromosome 6 of the radish reference genome; the polymorphism of the Indel-1 site is a deletion or insertion sequence, and the nucleotide sequence of the insertion sequence is shown in SEQ ID NO.1 in the sequence listing; 948 bases were added upstream of the Indel-1 site and 5 bases were added downstream to obtain the Indel-1 molecular marker, the nucleotide sequence of which is shown in SEQ ID NO.4 in the sequence table; The primers for amplifying the above-mentioned Indel-1 molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6 in the sequence listing.

[0031] Example 2 Detection of Indel-1 molecular marker in multiple radish varieties The 53 radish materials were identified using the Indel-1 molecular marker. The identification method was as follows: A. Extraction of genomic DNA from radish leaves Genomic DNA from radish leaves was extracted using a plant genomic DNA extraction kit (Solarbio).

[0032] B. PCR amplification PCR amplification was performed using primers labeled with Indel-1.

[0033] The PCR amplification reaction system was 20 μL, specifically: 2× Rapid Tap Master Mix 10 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, genomic DNA 1 μL, and ddH2O 7 μL.

[0034] The PCR amplification procedure is as follows: (1) 95°C, 3 min; (2) 95°C, 30 seconds; (3) 55°C, 30 seconds; (4) 72°C, 30 seconds; (5) Steps (2) to (4) were repeated 34 times; (6) 72°C, 5 min; (7) Store at 12℃.

[0035] C. PCR product detection The PCR amplification products were detected by agarose gel electrophoresis. After the electrophoresis, photos were taken and observed in the gel imaging system to determine the specific situation of the amplification products.

[0036] The electrophoresis results are as follows Figure 2-6 The results of the molecular marker identification were compared with the actual color of the fleshy roots of radish. The results showed that the agarose gel electrophoresis results of 50 of the 53 materials were consistent with the phenotype, with a coincidence rate of 94.3% (see Tables 6 and 7). It can be seen that the Indel-1 molecular marker can be used as a marker to distinguish the fleshy roots of radish materials as green, with high accuracy.

[0037] Table 6 Identification results of radish materials from 1 to 26

[0038] Table 7 Identification results of radish materials from 27 to 53

Claims

1. A molecular marker tightly linked to the green flesh trait of radish, characterized in that: The molecular marker is an Indel molecular marker, and the nucleotide sequence of the Indel molecular marker is shown in SEQ ID NO.4 in the sequence list; the primer for amplifying the molecular marker is used to detect the Indel site; the polymorphism of the Indel site is a deletion or insertion sequence, and the nucleotide sequence of the insertion sequence is shown in SEQ ID NO.1 in the sequence list.

2. The molecular marker tightly linked to the green flesh trait of radish according to claim 1, characterized in that: The Indel site is located at positions 45402470-45402485 of the full-length sequence of chromosome 6 of the radish reference genome.

3. The molecular marker tightly linked to the green flesh trait of radish according to claim 1, characterized in that: The primers for amplifying the molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.5 in the sequence list; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6 in the sequence list.

4. Use of primers for amplifying the molecular markers according to any one of claims 1 to 3 in identifying radish flesh traits.

5. The use according to claim 4, characterized in that: The radish flesh is green or white.

6. The use according to claim 4, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the radish leaves to be tested; (2) Using genomic DNA from radish leaves as a template, PCR amplification was performed using primers for amplifying molecular markers, and the PCR amplification products were detected; (3) If an amplification product with a length of 969 bp is amplified, and the nucleotide sequence of the amplification product is as shown in SEQ ID NO.4 in the sequence listing, the radish flesh trait to be detected is white flesh; if no amplification product is amplified, the radish flesh trait to be detected is green flesh.

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