Primer pair for amplification of eggplant fruit skin color gene

Abstract

The primer pair for amplifying eggplant fruit skin color gene is characterized in that the nucleotide sequence of the primer pair is as follows: the forward primer is Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; and the reverse primer is Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'. The detection result is more accurate, stable and reliable.

Description

Technical Field

[0001] This application belongs to the field of biomolecular detection and breeding, specifically involving primer pairs for amplifying the color gene of eggplant peel. Background Technology

[0002] eggplant( Solanum melongena Eggplant is an important vegetable crop widely cultivated in Asia and Africa. It boasts abundant germplasm resources and rich fruit shape polymorphism, making it an ideal material for studying fruit shape. Fruit shape is one of the main indicators of the commercial value of horticultural crops. Clarifying the genetic laws governing eggplant fruit shape can provide a basis for developing relevant molecular markers and breeding new varieties with fruit shapes preferred by consumers.

[0003] Anthocyanins are distributed in the vacuoles of plant cells, enabling certain plant organs to exhibit red, purple, blue, and other colors, and determining the flower color of most plants and the fruit color of some. Its basic structure is 3,5,7-hydroxy-2-phenylbenzopyran, and its precursor is phenylalanine. It is formed through a series of enzymatic reactions in the endoplasmic reticulum of the cytoplasm. Anthocyanin synthesis is regulated by both genetic and environmental factors. Genetic factors include structural genes and regulatory factors in the anthocyanin synthesis pathway. Environmental factors include light, temperature, exogenous hormones, and stress factors.

[0004] Domestic and international scholars have conducted extensive research on the localization of genes controlling the purple peel color in eggplants and the development of linkage markers. Regarding the localization of these genes, they are currently generally studied as a qualitative trait. Some studies have found that these genes are located on chromosomes 10 and 12 of the eggplant. In terms of linkage marker development, scholars both domestically and internationally have developed many molecular markers linked to these genes, but the degree of tightness of linkage and their applicability still need improvement. Summary of the Invention

[0005] The primer pair used for amplifying the eggplant peel color gene is characterized by the following nucleotide sequence: The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'.

[0006] Further: used for PCR amplification of the gene for purple peel in eggplant.

[0007] Further: used for amplification of the gene for green eggplant peel.

[0008] Further: used to identify genes responsible for eggplant peel color.

[0009] Further: Used to remove eggplant samples that do not contain the purple peel gene.

[0010] Further: Used to remove eggplant samples that do not contain the green peel gene.

[0011] Further: for selective breeding targeting eggplant skin color.

[0012] Beneficial effects: The primer pairs of this invention provide more accurate, stable, and reliable results for detecting eggplant peel color genes. Attached Figure Description

[0013] Figure 1 The amplification results are from Example 1. Note: A: sm524; B: sm518.

[0014] Figure 2 This is a photograph of sample sm524 from Example 1.

[0015] Figure 3 This is a photograph of sample sm518 from Example 1.

[0016] Figure 4 This is a sample table for Example 2.

[0017] Figure 5 This is the amplification result of Example 1. Specific Implementation

[0018] Example 1: Detection experiment of the purple peel gene of eggplant. Eggplant genomic DNA was extracted using a modified CTAB method. 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. Step 2: Genotyping the above-mentioned test materials, using the genomic DNA of the test materials as the template; Step 2.1: Perform PCR amplification 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. The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'.

[0019] 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. 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. Get as Figure 1 The amplification results are shown below; Step 2.2: Sequencing of the 199bp fragment, its DNA sequence is as follows: CTCGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG The 193bp fragment was sequenced, and its DNA sequence is as follows: CTTGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG Example 2: Detection of genes in purple-skinned eggplant. 1. Targeting the construction of the target audience This experiment used the purple-skinned inbred line sm524 (characterized by purple pericarp) as the female parent and the non-purple-skinned inbred line sm518 (characterized by green pericarp and flowers) as the male parent to construct an F2 population. An F2 population of 300 individual plants was obtained, all of which were either purple or non-purple. Eggplant genomic DNA was extracted using a modified CTAB method.

[0020] 2. Molecular marker development Based on the published eggplant genome nucleotide sequence, primers were first designed and synthesized at InDel sites with a difference greater than 3 bp between the two parents. The polymorphism of these sites was verified using DNA from both parents, and an Indel marker was developed on chromosome 12.

[0021] 3. Obtaining the molecular marker Wz-1 Polymorphic screening revealed that the molecular marker Wz-1 exhibits stable polymorphism between the parents. Based on the detection results, Wz-1 was determined to be a co-dominant marker, and the amplified bands produced by this marker were clear and showed significant differences between the parents. Figure 1 ). Figure 1 This indicates that the molecular marker Wz-1 contains a 199 bp fragment in the genomic DNA amplification product of sm524 and a 193 bp fragment in the genomic DNA amplification product of sm518. The primer nucleotide sequence for marker Wz-1 is shown below: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; Wz-1R: 5'- CCTATCACACCTAATTGTTAAACTG-3'.

[0022] The 199bp fragment contained in sm524: CTCGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG The 193bp fragment contained in sm518: CTTGTCAAAGACTGATTACACGATAATAAACCAAAAAACATAAAAAAACTTTAGACCTCATACACCCAACTACTTGTGACGTGTGATTTGCAGTGGCTATTTTAGGCTGAAATTAAAAGGTTATTAAGTTAGTTTAAAAAAAAAAAAAACTTGAAAAGTAAGGTATGGTCAGTTTAACAATTAGGTGTGATAGG 4. Genetic linkage analysis The genotypes of 300 individuals from the F2 population were analyzed using the polymorphic molecular marker Wz-1 obtained through screening. Combined with the field fertility assessment results of these 300 individuals, the linkage relationship between Wz-1 and the purple peel trait was calculated using JoinMap 4.0 software. The results showed that the genetic distance between the molecular marker Wz-1 and the gene controlling purple peel was 0.2 cM.

[0023] Example 3: Detection of the purple gene in the fruit peel 1. Test materials 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.

[0024] 2. Test Methods Genotyping was performed on the above-mentioned test materials, using the genomic DNA of the test materials as the template.

[0025] 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.

[0026] 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. 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.

[0027] 3. Test Results Electrophoresis results as follows Figure 5 As 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. 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. 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. Primer pairs for amplifying the color gene of eggplant peel, characterized in that: The nucleotide sequences of the primer pair are as follows: The forward primer is: Wz-1F: 5'-CTTGTCAAAGACTGATTACACGATA-3'; The reverse primer is: Wz-1R: 5'-CCTATCACACCTAATTGTTAAACTG-3'.

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