Method for identifying eggplant purple fruit skin gene

Abstract

The method for identifying eggplant purple peel gene is characterized in that: step 1, preparing the test material; step 2, performing PCR amplification on the test material, the nucleotide sequence of the primer pair is as follows: the forward primer is Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; the reverse primer is Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'; whether the amplification result contains a 199bp fragment is observed, if yes, it indicates that the test material contains the purple peel gene, and if not, it indicates that the test material does not contain the purple peel gene. The detection result of the purple peel gene in the application is more accurate, stable and reliable.

Description

Technical Field

[0001] This application belongs to the field of biomolecular detection and breeding, specifically involving a method for identifying the gene for purple peel in eggplant. Background Technology

[0002] eggplant( Solanum melongena It is a vegetable widely grown around the world and loved by people all over the world;

[0003] The color of eggplant peel influences consumers' purchasing desire, and therefore deserves in-depth research.

[0004] Anthocyanin synthesis directly affects the color of eggplant peel and calyx. Eggplant genes influence the distribution and expression of anthocyanins in the vacuoles of plant cells, but they are not the sole determinant of fruit color. Environmental factors can also affect anthocyanin synthesis, so relying solely on the peel color of samples for seed selection is not entirely reliable. Identifying eggplant peel color genes can provide a rigorous scientific basis for eggplant fruit breeding and selection.

[0005] Domestic and foreign scholars have conducted extensive research on controlling the gene for purple peel in eggplants; however, there is room for improvement in the accuracy and reliability of purple peel gene detection. Summary of the Invention

[0006] A method for identifying the gene for purple peel in eggplant, characterized by:

[0007] Step 1: Prepare the sample;

[0008] Step 2: Perform PCR amplification on the sample. The nucleotide sequences of the primer pairs are as follows:

[0009] The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3';

[0010] The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3';

[0011] Step 3: Observe whether the amplification result contains a 199bp fragment. If it does not, it means that the sample does not contain the purple peel gene.

[0012] Further, in step 4, the 199bp fragment is sequenced. If the DNA sequence of the 199bp fragment is:

[0013] The result of the code CTCGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG indicates that the sample contains the purple fruit peel gene.

[0014] Further, the PCR reaction system (12 μl).

[0015] Furthermore, in the PCR reaction system, there is 2 μl of DNA working solution.

[0016] Furthermore, in the PCR reaction system, 2xEs TaqMasterMix 5ul.

[0017] Furthermore, in the PCR reaction system, there are 1 μl (10 uM / L) of forward primer and 1 μl (10 uM / L) of reverse primer.

[0018] Furthermore, in the PCR reaction system, the total volume was brought up to 12 μl with 3 μl ddH2O.

[0019] Beneficial effects: The detection results of the purple peel gene in this invention are more accurate, more stable, and more reliable. Attached Figure Description

[0020] Figure 1 The amplification results are from Example 1. Note: A: sm524; B: sm518.

[0021] Figure 2 This is a photograph of sample sm524 from Example 1.

[0022] Figure 3 This is a photograph of sample sm518 from Example 1.

[0023] Figure 4 This is a sample table for Example 2.

[0024] Figure 5 This is the amplification result of Example 2. Specific Implementation

[0025] Example 1: Detection experiment of the purple peel gene of eggplant. Eggplant genomic DNA was extracted using a modified CTAB method.

[0026] Step 1: Obtain purplish-red eggplant sm524 and variegated green eggplant sm518 as experimental materials. The samples can be obtained from the Vegetable Research Institute of Guangxi Academy of Agricultural Sciences.

[0027] Step 2: Genotyping the above-mentioned test materials, using the genomic DNA of the test materials as the template;

[0028] Step 3: Perform PCR amplification

[0029] The PCR reaction system (12 μl) consisted of: 2 μl of DNA working solution, 5 μl of 2xEs TaqMasterMix, 1 μl each of forward and reverse primers (10 μM / L), and 3 μl of ddH2O to bring the total volume to 12 μl.

[0030] The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3';

[0031] The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'.

[0032] PCR amplification reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0033] The amplification products were subjected to 8% non-denaturing polyacrylamide gel electrophoresis, stained with silver nitrate, and then observed and photographed. The PCR amplification products were also sequenced.

[0034] Get as Figure 1 The amplification results are shown below;

[0035] The 199bp fragment was sequenced, and its DNA sequence is as follows:

[0036] CTCGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAAGTTTAAAAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG

[0037] The 193bp fragment was sequenced, and its DNA sequence is as follows:

[0038] CTTGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG

[0039] Example 2: Detection of the purple gene in the fruit peel

[0040] 1. Test materials

[0041] This invention utilizes, for example Figure 4 The molecular marker Wz-1 was validated in the eight purple-skinned eggplant inbred lines and varieties and the eight non-purple-skinned eggplant inbred lines and varieties shown. The inbred lines and varieties used were obtained from the Vegetable Research Institute of Guangxi Academy of Agricultural Sciences.

[0042] 2. Test Methods

[0043] Genotyping was performed on the above-mentioned test materials, using the genomic DNA of the test materials as the template.

[0044] The PCR reaction system (12 μl) consisted of: 2 μl of DNA working solution, 5 μl of 2xEs TaqMasterMix, 1 μl each of forward and reverse primers (10 μM / L), and 3 μl of ddH2O to bring the total volume to 12 μl.

[0045] PCR amplification reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0046] The amplification products were subjected to 8% non-denaturing polyacrylamide gel electrophoresis, stained with silver nitrate, and then observed and photographed. The PCR amplification products were also sequenced.

[0047] 3. Test Results

[0048] Electrophoresis results as follows Figure 5As shown, lanes 1: sm524; lane 2: sm167; lane 3: sm623; lane 4: sm005; lane 5: sm047; lane 6: sm011; lane 7: sm021; lane 8: sm613; lane 9: sm006; lane 10: sm279; lane 11: sm503; lane 12: sm544; lane 13: sm545; lane 14: sm530; lane 15: sm518; lane 16: sm546. The fruit peel color phenotypes corresponding to the verification materials are shown in Table 1. Lanes 1-10 are purple-skinned materials, and lanes 11-20 are non-purple-skinned materials. The PCR amplification products of molecular marker Wz-1 in purple-skinned varieties (sm524; sm167; sm623; sm005; sm047; sm011; sm021; sm613) all contained a 199bp fragment and did not contain a 193bp fragment.

[0049] The PCR amplification products of the molecular marker Wz-1 in non-purple-skinned varieties (sm006; sm279; sm503; sm544; sm545; sm530; sm518; sm546) all contained a 193bp fragment and did not contain a 199bp fragment. The genotype and fruit color phenotype identification results of the tested varieties were consistent, indicating that the molecular marker Wz-1 of the present invention has strong specificity and can be used for the identification and screening of eggplant fruit color phenotype.

Claims

1. A method for identifying the gene for purple peel in eggplant, characterized by: Step 1: Prepare the sample; Step 2: Perform PCR amplification on the sample. The nucleotide sequences of the primer pairs are as follows: The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'; Step 3: Observe whether the amplification result contains a 199bp fragment. If it does not, it means that the sample does not contain the purple peel gene.

2. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: Step 4: Sequencing the 199bp fragment. If the DNA sequence of the 199bp fragment is: The result of the code CTCGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG indicates that the sample contains the purple fruit peel gene.

3. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: The PCR reaction volume was 12 μl.

4. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: In the PCR reaction system, there is 2 μl of DNA working solution.

5. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: In the PCR reaction system, there are 5 μL of 2xEs TaqMasterMix.

6. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: In the PCR reaction system, there are 1 μl of forward primer and 1 μl of reverse primer.

7. The method for identifying the purple peel gene of eggplant as described in claim 1, characterized in that: In the PCR reaction system, the total volume was brought up to 12 μl with 3 μl ddH2O.

Images