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Two color-related mutations in apple MdMYB1 gene and detection method thereof

An apple and gene technology, applied in the biological field, can solve problems such as inability to detect, and achieve the effects of reduced experimental cost, simple and practical identification method, and shortened electrophoresis time.

Inactive Publication Date: 2011-11-16
SHANDONG INST OF POMOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the dCAPS marker is that the length of the fragment after digestion is only 29bp, which cannot be detected by ordinary agarose electrophoresis, and the expensive NuSieve GTG agarose with a price of about 7000 yuan / 125g is required for electrophoresis detection

Method used

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  • Two color-related mutations in apple MdMYB1 gene and detection method thereof
  • Two color-related mutations in apple MdMYB1 gene and detection method thereof
  • Two color-related mutations in apple MdMYB1 gene and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Detection of mutations and identification of plant coloring traits of apple cultivars by CAPS marker method

[0031] Step 1, test materials: leaves or flowers of 8 common apple varieties: Fuji, Gala, Jinshuai, Guoguang, Hongxing, Hongyu, India, and Green Banana.

[0032] Step 2, genomic DNA extraction: same as the aforementioned sequencing method.

[0033] Step 3, PCR amplification: same as the aforementioned sequencing method.

[0034] Step 4, enzyme digestion experiment: Take 5 μL of the PCR product and add it to a test tube, then add 2 μL of reaction buffer (10x), 1 μL (10 U) of restriction endonuclease Pml I and 12 μL of water, and carry out enzyme digestion in a 37°C water bath for 2-3 hours. Mix 20 μL of digested products with 5 μL of loading dye, stain with ethidium bromide, and perform electrophoresis on 1.2% agarose gel at 120V to check the digested products. The results are as follows: image 3 shown.

[0035] Step 5, analysis of coloring trait...

Embodiment 2

[0036] Example 2: Detection of mutations and identification of coloring traits of apple hybrid offspring by CAPS marker method

[0037] Step 1, test material: use Fuji and Gala apples as parents to cross to obtain seeds, and the leaves of apple hybrid progeny plants obtained after the seeds are sown are used as test materials.

[0038] Step 2, genomic DNA extraction: same as the aforementioned sequencing method.

[0039] Step 3, PCR amplification: same as the aforementioned sequencing method.

[0040] Step 4, enzyme digestion experiment: Take 5 μL of the PCR product and add it to a test tube, then add 2 μL of reaction buffer (10x), 1 μL (10 U) of restriction endonuclease Pml I and 12 μL of water, and carry out enzyme digestion in a 37°C water bath for 2-3 hours. 20 μL of digested products were mixed with 5 μL of loading dye, stained with ethidium bromide, and checked by electrophoresis on 1.2% agarose gel at 120V.

[0041] Step 5, analysis of coloring traits of apple hybri...

Embodiment 3

[0042] Example 3: Using Mutation Site 206 to Identify Plant Coloring Traits of Apple Varieties

[0043] Step 1, test material: the same as the aforementioned sequencing method.

[0044] Step 2, genomic DNA extraction: same as the aforementioned sequencing method.

[0045] Step 3, PCR amplification: same as the aforementioned sequencing method.

[0046] Step 4, electrophoresis inspection: the same as the aforementioned sequencing method.

[0047] Step 5, PCR product sequencing: same as the aforementioned sequencing method.

[0048] Step 6, analysis of coloring traits of apple cultivars: In view of the fact that the newly found mutation site 206 (that is, at -1624bp upstream of the transcription start point of MdMYB1-1 gene) can provide the same mutation site as the former apple coloring traits dCAPS molecular marker mutation site The information shows that the base information of the newly obtained mutation site 206 is the same as the previous apple coloring traits dCAPS m...

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Abstract

Two color-related mutations in apple MdMYB1 gene and a detection method thereof. The invention belongs to the field of biotechnology and provides a low-cost molecular breeding means for the improvement of apple color traits. A pair of primers is designed by the use of apple color related gene MdMYB1, and apple genome DNA is taken as a template to carry out PCR amplification so as to obtain a PCR amplification segment. Sequence analysis is carried out on PCR amplification segments of 8 apple varieties to find out two gene mutations related with the apple color traits, wherein one gene mutationis Pm1 Iendonuclease site. The enzyme site is used to develop a CAPS marker Mb2P for detecting the mutation. The substantial progress lies in that common agarose gel electrophoresis can be used for the detection of the developed marker so as to greatly reduce experimental cost, in comparison with expensive NuSieve GTG agarose gel electrophoresis which is needed for the detection of the dCAPS marker in the prior art. By the use of the CAPS marker or the sequencing method to detect mutations, apple varieties and the color traits of hybrid posterity fruits can be identified. The color traits of hybrid posterity apples are selected at an early stage to abandon parts of plants, so as to save the management and screening cost of progeny plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the relationship between two mutations in the apple MdMYB1 gene and the coloring properties of apple fruit and a mutation detection method. Background technique [0002] Apple (Malus domestica Borkh.) is an important bulk fruit in my country's fruit tree industry. The coloring problem is an important factor affecting the quality of apples in many production areas in my country. It is urgent to cultivate varieties with good coloring. By studying the sequence variation of apple coloring-related genes, developing molecular markers for fruit coloring traits, early identification of fruit coloring traits of hybrid offspring plants, and early screening of hybrid offspring plants will help reduce the cost and workload of early trials Cultivate new varieties of apples with good coloring as soon as possible, and enhance the quality and market competitiveness of my country's apples....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 苑克俊黄立香魏海蓉刘庆忠
Owner SHANDONG INST OF POMOLOGY
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