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Three mutations of FPPS gene of Malus domestica Borkh. and identification method thereof

A technology of apples and genes, applied in the biological field, can solve the problems of finding alleles and base mutation research reports, etc., and achieve the effect of saving management and screening costs and saving costs

Inactive Publication Date: 2011-04-13
SHANDONG INST OF POMOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for another key enzyme gene (apple farnesyl pyrophosphate synthase gene FPPS) in the α-farnesene synthesis pathway, only one cDNA sequence of FPPS gene is found in GenBank. We have an article prone to tiger skin disease Research reports on the promoter and 5'UTR sequence of the FPPS gene in apple varieties (Yuan Kejun et al., 2009), but no research reports on alleles and base mutations related to apple tiger skin disease traits have been found

Method used

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  • Three mutations of FPPS gene of Malus domestica Borkh. and identification method thereof
  • Three mutations of FPPS gene of Malus domestica Borkh. and identification method thereof
  • Three mutations of FPPS gene of Malus domestica Borkh. and identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Specific primer molecular labeling method to detect mutations and identify apple varieties or fruit tiger skin disease traits of hybrid offspring

[0048] Step 1, test materials: leaves or flowers of four apple varieties, Guoguang, Green Banana, Golden Delicious and Royal Gala, among which Royal Gala is a hybrid offspring of Jinguan.

[0049] Step 2, genomic DNA extraction: Immediately after grinding the test material, add 0.6mL 2% CTAB extraction buffer [2% CTAB, 100mmol / L Tris-HCl (pH8.0), 20mmol / L EDTA, 1.4 mol / L NaCl, 2% PVP-40, 2% β-mercaptoethanol], extracted in a 65°C water bath for 30min, during which time the sample tube was gently inverted 3 times. Take out and add 0.6mL of chloroform:isoamyl alcohol (24:1), turn over gently, mix well, and centrifuge at 12000rpm for 10min. Transfer the supernatant to another new test tube, add an equal volume of isopropanol pre-cooled at -20°C, shake gently, centrifuge at 12000rpm for 10min, and remove the supern...

Embodiment 2

[0053] Example 2: Using the sequencing method to obtain the base information of the mutation site to identify the tiger skin disease traits of apple varieties or hybrid progeny

[0054] Step 1, test material: leaves or flowers of five apple varieties: Fuji, Guoguang, Green Banana, Red Star, and Golden Delicious, among which Fuji is a hybrid offspring of Guoguang.

[0055] Step 2, genomic DNA extraction: the same as the aforementioned molecular marker method.

[0056] Step 3, PCR amplification: perform PCR amplification with f3x as forward primer and f4x as reverse primer. The PCR reaction solution (20 μL) contained 0.5 μL (25 ng) of genomic DNA and 19.5 μL of reaction mixture, and one part of the mixture consisted of 0.4 μL of dNTPs (10 mmol / L), 2 μL of 10x Taq buffer, 0.5 μL of forward primer (10 μmol / L), 0.5 μL of reverse primer (10 μmol / L), 0.25 μL (5 U) of Taq DNA polymerase and 15.85 μL of water. The PCR reaction was performed for 35 cycles, each cycle including denat...

Embodiment 3

[0061] Example 3: Using CAPS molecular markers to detect mutations and identify tiger skin disease traits in apple varieties

[0062] Step 1, test materials: leaves or flowers of six common apple varieties: Golden Delicious, Royal Gala, Fuji, Guoguang, Red Star, and Green Banana.

[0063] Step 2, genomic DNA extraction: the same as the aforementioned molecular marker method.

[0064] Step 3, PCR amplification: perform PCR amplification with f3x as forward primer and f4x as reverse primer. The PCR reaction solution (20 μL) contained 0.5 μL (25 ng) of genomic DNA and 19.5 μL of reaction mixture, and one part of the mixture consisted of 0.4 μL of dNTPs (10 mmol / L), 2 μL of 10x Taq buffer, 0.5 μL of forward primer (10 μmol / L), 0.5 μL of reverse primer (10 μmol / L), 0.25 μL (5 U) of Taq DNA polymerase and 15.85 μL of water. The PCR reaction was performed for 35 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 56°C for 1 min and primer extension at 72°C fo...

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Abstract

The invention relates to three mutations of a farnesyl diphosphate synthase (FPPS) gene of Malus domestica Borkh. and an identification method thereof, which belong to the field of biotechnology and provide a simple, practicable and cost-saving molecular marker breeding preselection method for breeding a superficial scald-resistant Malus domestica Borkh. variety. The identification method comprises the following steps of: designing a primer by using the sequence of the FPPS gene of the Malus domestica Borkh. and performing polymerase chain reaction (PCR) amplification by using a genome deoxyribose nucleic acid (DNA) of the Malus domestica Borkh. to obtain a PCR amplification segment; and sequencing and analyzing the PCR amplification segment of the superficial scald-resistant Malus domestica Borkh. variety and a superficial scald-susceptible Malus domestica Borkh. variety to find out genetic mutations related to characters of a superficial scald of the Malus domestica Borkh. at three positions, namely -262bp, -434bp and -600bp at the upstream of a transcription start point of the FPPS gene. By detecting the mutations through a molecular marker or sequencing method, the Malus domestica Borkh. variety and the characters of the superficial scald of fruits of a filial generation can be identified, and the characters of the superficial scald of the fruits of the filial generation of the Malus domestica Borkh. can be selected at an early stage so as to save cost for managing and screening progeny plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the relationship between three mutations in the promoter region of apple FPPS gene and the character of apple tiger skin disease, the sequence of FPPS allelic gene and the mutation identification method. Background technique [0002] Apple (Malus domestica Borkh.) is an important bulk fruit in my country's fruit tree industry. Tiger skin disease is an important physiological disease of apples and pears during storage, which seriously affects the appearance and commercial value of fruits. The occurrence of tiger skin disease is closely related to α-farnesene (Hu Xiaosong et al., 2002). The synthesis of apple α-farnesene involves 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, 3-hydroxy-3-methylglutaryl coenzyme A), farnesyl pyrophosphate synthase (FPPS, farnesyl diphosphate synthase) and α-farnesene synthase (AFS, alpha-farnesenesynthase) and other enzyme genes (R...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/52
Inventor 苑克俊刘庆忠艾呈祥魏海蓉
Owner SHANDONG INST OF POMOLOGY
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