A molecular marker closely linked to the green flesh trait of radish and its application

Abstract

The present invention provides a molecular marker closely linked to the green flesh trait of radish and its application, belonging 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 shown as SEQ ID NO.4 in the sequence listing; the primer for amplifying the molecular marker is used to detect the Indel locus; the polymorphism of the Indel locus is a deleted or inserted sequence, and the nucleotide sequence of the inserted sequence is shown as SEQ ID NO.1 in the sequence listing. Identification was carried out on 53 radish materials using the primer for amplifying the Indel molecular marker of the present invention, and the coincidence rate between the identification result and the phenotype of radish reached 94.3%.

Description

Technical Field

[0001] The present invention relates to a molecular marker closely linked to the green flesh trait of radish and its application, belonging to the technical field of nucleic acid-containing assay methods. Background Art

[0002] Radish is a popular vegetable with a long cultivation history in China. The flesh color of its fleshy root can be white, green, red, etc. Radishes with different flesh colors vary in taste, uses, and nutritional value. For example, white-fleshed radishes have a slightly sweet taste and are suitable for stewing; green-fleshed radishes are crispy, sweet, and juicy and are suitable for raw consumption; red-fleshed radishes are rich in anthocyanins and have relatively high nutritional value.

[0003] The main method for identifying the flesh color of radish is to cut open the fleshy root of the radish at the mature stage and then conduct phenotypic identification. This belongs to a destructive detection method, and it requires a large amount of time and manpower, with low efficiency. Moreover, the identification result is easily affected by personal experience and has strong subjectivity. By using molecular marker detection, the flesh color of radish can be screened at an early stage, shortening the breeding cycle, reducing costs, and the results are more stable and reliable. More importantly, it will not cause damage to the radish, can maintain the integrity of the radish, and is more conducive to the survival of the mother plants and the screening of commercial radishes.

[0004] There are various types of molecular markers. Indel (Insertion-Deletion) molecular marker is one of the common markers. Indel molecular marker, namely insertion-deletion marker, refers to the polymorphism 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 root, which is used to distinguish the red flesh and white flesh traits of radish; CN116875737A discloses a primer group for identifying the flesh color trait of radish fleshy root 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 radish fleshy root in the prior art, there is no report on identifying the green flesh trait of radish. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention provides a molecular marker closely linked to the green flesh trait of radish and its application, achieving the following invention objectives: the green flesh trait of radish can be detected, and the accuracy rate of the detection result is high.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A molecular marker closely linked to the green flesh trait of radish, the molecular marker being an Indel molecular marker, and the nucleotide sequence of the Indel molecular marker being as shown in SEQ ID NO.4 in the sequence listing; the primer for amplifying the molecular marker is used to detect the Indel locus; the polymorphism of the Indel locus is a deletion or insertion sequence, and the nucleotide sequence of the insertion sequence is as shown in SEQ ID NO.1 in the sequence listing.

[0010] The Indel locus is located at positions 45402470 - 45402485 of the full-length sequence of chromosome 6 of the radish reference genome.

[0011] The primer for amplifying the molecular marker includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6 in the sequence listing.

[0012] Application of the primer for amplifying the molecular marker in identifying the flesh trait of radish.

[0013] The radish flesh trait is green flesh or white flesh.

[0014] The said application includes the following steps:

[0015] (1) Extract the genomic DNA of the radish leaves to be detected;

[0016] (2) Using the genomic DNA of the radish leaves as a template, perform PCR amplification with the primer for amplifying the molecular marker, and detect the PCR amplification product;

[0017] (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, then the flesh trait of the radish to be detected is white flesh; if no amplification product is amplified, then the flesh trait of the radish to be detected is green flesh.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention screens an Indel locus closely linked to the green flesh trait of radish, designs an Indel molecular marker and a primer for amplifying the molecular marker based on this locus, and uses this primer to amplify in the radish DNA to be detected. The green flesh trait of radish can be identified according to the situation of the amplification product, with high accuracy.

[0020] When the primer for amplifying the Indel molecular marker of the present invention is used for identification in 53 radish materials, the coincidence rate between the identification result and the phenotype of radish reaches 94.3%. Description of the Drawings

[0021] Figure 1 It is the distribution map of Indel sites on 9 chromosomes of the radish reference genome;

[0022] Figure 2 It is the amplification electrophoresis map of Indel-1 marker in radishes No. 1-6;

[0023] Among them, No. 1-3 are green-fleshed radishes, and No. 4-6 are white-fleshed radishes;

[0024] Figure 3 It is the amplification electrophoresis map of Indel-1 marker in radishes No. 7-11;

[0025] Among them, No. 7-10 are green-fleshed radishes, and No. 11 is a white-fleshed radish;

[0026] Figure 4 It is the amplification electrophoresis map of Indel-1 marker in radishes No. 12-26;

[0027] Among them, No. 12-18 are green-fleshed radishes, and No. 19-26 are white-fleshed radishes;

[0028] Figure 5 It is the amplification electrophoresis map of Indel-1 marker in radishes No. 27-39;

[0029] Among them, No. 27-30 are green-fleshed radishes, and No. 31-39 are white-fleshed radishes;

[0030] Figure 6 It is the amplification electrophoresis map of Indel-1 marker in radishes No. 40-53;

[0031] Among them, No. 40-50 are green-fleshed radishes, and No. 51-53 are white-fleshed radishes. Detailed implementation manners

[0032] Example 1 Development of Indel molecular markers

[0033] 1. Indel identification

[0034] 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 1-4. Among them, there are 187 white-fleshed radishes, 53 green-fleshed radishes, 12 red-fleshed radishes, and 38 purple-fleshed radishes. The above materials are collected and preserved by the National Germplasm Resources Bank of the Chinese Academy of Agricultural Sciences, Weifang University, and Weifang Academy of Agricultural Sciences.

[0035] Table 1 Number information of the 1st - 76th radish materials

[0036]

[0037] Table 2 Number information of the 77th - 152nd radish materials

[0038]

[0039] Table 3 Serial number information of the 153 - 228th radish materials

[0040]

[0041] Table 4 Serial number information of the 229 - 290th radish materials

[0042]

[0043] The above 290 materials were re - sequenced on the illumina Novaseq 6000 sequencing platform, and a total of 1597 GB of high - quality base data was obtained. The average sequencing depth was 12.96x, and the average Q30 was 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 the Gatk4 program was used to identify Indels.

[0044] Combined with the VCF tools software, the Indels were filtered and quality - controlled, and the 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 integrity was greater than 80%; ④ Only biallelic genes were retained. Finally, a total of 308,324 Indel sites were obtained, which were distributed on 9 chromosomes of the radish reference genome ( Figure 1 ).

[0045] 2. GWAS analysis of the green - flesh trait of radish

[0046] Using the Tassel 5.0 software with the GLM + PCA model, combined with the flesh - color trait of the radish fleshy root, a genome - wide association analysis was carried out, and the P - value threshold was set to 1×10 -7 , and finally 1697 Indel sites significantly associated with the green - flesh trait were obtained.

[0047] 3. Screening of Indel sites

[0048] The above 1697 Indel sites were analyzed one by one, and the Indel sites that met the following criteria were selected: ① The length of the Indel fragment was ≥6 bp; ② The coincidence rate of the Indel genotype and the radish green / white flesh color phenotype was ≥90%.

[0049] Finally, 8 Indel sites were screened (see Table 5).

[0050] Table 5 8 Indel sites significantly related to the green - flesh trait of radish

[0051]

[0052] According to the physical positions of the Indel loci, DNA sequences of 1000 bp each 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 with the primers of the above-mentioned Indel loci. The results showed that only the Indel-1 locus could well distinguish the green-fleshed and white-fleshed traits of radishes; the other Indel-2 to Indel-8 could not well distinguish the green-fleshed and white-fleshed traits of radishes.

[0053] Finally, the Indel-1 locus was selected as the Indel locus tightly linked to the green-fleshed trait of radishes.

[0054] 4. Development of Molecular Markers and Primers

[0055] The Indel-1 locus 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 locus is a deletion or insertion sequence, and the nucleotide sequence of the insertion sequence is as shown in SEQ ID NO.1 in the sequence listing;

[0056] 948 bases were added upstream and 5 bases were added downstream of the Indel-1 locus to obtain the Indel-1 molecular marker, and its nucleotide sequence is as shown in SEQ ID NO.4 in the sequence listing;

[0057] The primers for amplifying the above Indel-1 molecular marker include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6 in the sequence listing.

[0058] Example 2 Detection of the Indel-1 Molecular Marker in Multiple Radish Cultivar Materials

[0059] Fifty-three radish materials were identified using the Indel-1 molecular marker. The identification method was as follows:

[0060] A. Extraction of Genomic DNA from Radish Leaves

[0061] The genomic DNA of radish leaves was extracted using a plant genomic DNA extraction kit (Solarbio).

[0062] B. PCR Amplification

[0063] PCR amplification was performed using the primers of the Indel-1 molecular marker.

[0064] 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.

[0065] The PCR amplification procedure is as follows:

[0066] (1) 95°C, 3 min;

[0067] (2) 95°C, 30 seconds;

[0068] (3) 55°C, 30 seconds;

[0069] (4) 72°C, 30 seconds;

[0070] (5) Steps (2) to (4) were repeated 34 times;

[0071] (6) 72°C, 5 min;

[0072] (7) Store at 12℃.

[0073] C. PCR product detection

[0074] 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.

[0075] The electrophoresis results are as follows Figures 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.

[0076] Table 6 Identification results of radish materials from 1 to 26

[0077]

[0078] Table 7 Identification results of radish materials from 27 to 53

[0079]

Claims

1. A molecular marker closely 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 as shown in SEQ ID No.

1. The Indel molecular marker is located at positions 45402470-45402485 of the full-length sequence of chromosome 6 of the radish reference genome.

2. 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 as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6 in the sequence listing.

3. Use of a primer for amplifying the molecular marker according to claim 1 in identifying the flesh traits of radish, characterized in that: The radish pulp trait is green pulp or white pulp. The primers for amplifying the molecular marker according to claim 1 include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6 in the sequence listing.

4. The application according to claim 3, characterized in that: It includes the following steps: (1) Extract the genomic DNA of the radish leaves to be detected; (2) Using the genomic DNA of the radish leaves as a template, perform PCR amplification with the primers for amplifying the molecular marker, and detect the PCR amplification product; (3) If an amplification product with a length of 969bp is amplified, and the nucleotide sequence of the amplification product is as shown in SEQ IDNO.4 in the sequence listing, then the radish pulp trait to be detected is white pulp; if no amplification product is amplified, then the radish pulp trait to be detected is green pulp.

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