High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker

A technology for melon and pulp, applied in the field of molecular biology, can solve problems such as cumbersome processes, achieve high analytical throughput, reduce human errors, and speed up transformation

Inactive Publication Date: 2015-12-30
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tzuri et al. (2015) found the CmOr gene that controls the color traits of melon pulp based on the results of previous studies on the location of genes related to flesh color and the genome information of melon. The cloning of the gene CmOr that controls the color of melon flesh enables the development of functional molecules based on the target gene sequence. However, the development of CAP

Method used

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  • High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker
  • High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker
  • High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. High-throughput KASP marker design of the gene CmOr related to the color and shape of melon pulp and development of its special primer sequence

[0043] 1. Extraction of tested muskmelon materials and their genomic DNA

[0044] A. Selection of test materials

[0045] Test material: orange-red flesh melon M285 (A:A genotype), green-flesh melon Lubao (G:G genotype), and white-flesh melon Elizabeth (G:G genotype) with known genotypes of the CmOr gene. Among them, the genotypes of the CmOr genes of these three melons have been sequenced and verified.

[0046] B. Genomic DNA extraction

[0047] The test materials were subjected to DNA extraction treatment respectively to obtain the genomic DNA of the test materials;

[0048] DNA extraction was improved on the basis of the method (MurrayM, ThompsonWF.RapidisolationofhighmolecularweightplantDNA[J].NuclAcidRes,1980,8:668-673.) with reference to Murray et al. (1980); the specific steps are as follows:

[0049] (1)...

Embodiment 2

[0078] Example 2, the establishment of a method for detecting the CmOr gene of muskmelon using high-throughput KASP markers

[0079] 1. Genomic DNA extraction

[0080] See Step 1 of Example 1.

[0081] 2. PCR amplification

[0082] Using the genomic DNA extracted in step 1 as a template, the special primers developed in Example 1 for detecting the KASP marker of the melon CmOr gene were used for PCR amplification respectively to obtain PCR amplification products.

[0083] KASP genotyping PCR reaction system:

[0084] 96-well plate: 10ng genomic DNA, 5μl KASPV4.02×MasterMix, 0.14μl KASP72×assaymix, add ddH 2 0 to 10 μl.

[0085] 384-well plate: 5ngDNA, 2.5μl KASPV4.02×MasterMix, 0.07μl KASP72×assaymix, add ddH 2 0 to 5 μl.

[0086] 1536-well plate: 5ngDNA, 2.5μl KASPV4.02×MasterMix, 0.07μl KASP72×assaymix, add ddH 2 0 to 5 μl.

[0087] Among them, KASPV4.02×MasterMix is ​​a product of LGC Company, and the catalog number of KASPV4.02×MasterMix for 96 / 384-well plates is K...

Embodiment 3

[0098] Example 3. Verification of the high-throughput KASP marker detection of the CmOr gene in melon and its application in breeding

[0099] 1. Test materials

[0100] The test materials include: the orange-red flesh melon variety M285 (A:A genotype) is used as the donor parent, and the white-flesh melon variety Elizabeth (G:G genotype) is used as the recurrent parent, and the color traits of the melon flesh are backcrossed. M285 was crossed with Elizabeth to obtain the corresponding F1 generation, the F1 generation was self-crossed to obtain the F2 population, and the F1 was backcrossed with the recurrent parent Elizabeth to obtain the BC population. The orange-red flesh melon varieties PI414723 (A: A genotype) and Védrantais (A: A genotype), the green flesh melon variety Lubao (G: G genotype), the parents Elizabeth (G: G genotype) and M285 (A : A genotype) as a control for molecular marker-assisted marker screening. The genotype of the CmOr gene of the control melon has ...

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Abstract

The invention discloses a high-flux molecular marker for identifying pulp color of Cucumis melo L. and a special primer for the high-flux molecular marker. The special primer disclosed by the invention is used for detecting a KASP primer for identifying a CmOr gene of the Cucumis melo L., and the specific nucleotide sequences of the specific primer are show in sequences 1 to 3 in a sequence table. A high-flux molecular marker system, based on a PCR (Polymerase Chain Reaction) SNP (Single Nucleotide Polymorphism) Line platform, which is provided by the invention is used for detecting a CmOr genetype of the Cucumis melo L., the operation flow is fully automatic, and artificial errors are reduced; the high-flux molecular marker is high in analysis flux and is very suitable for simultaneously detecting a large amount of samples. The molecular marker for high-flux detection is designed on the basis of a sequence of a color control gene CmOr of Cucumis melo L. and is applied to transformation breeding of color characters of the Cucumis melo L., so that the time and the labor cost can be greatly saved, the breeding efficiency of assisted selection of the molecular marker is improved, and transformation breeding of the pulp color of the Cucumis melo L. to an excellent skeleton inbred line is accelerated.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a high-throughput molecular marker and a special primer for identifying the color of muskmelon flesh. Background technique [0002] Melon (Cucumismelo L.) is an important economic crop of Cucurbitaceae Cucumber. The mesocarp and endocarp, the main edible parts of melons, are collectively referred to as the pulp. The color of the flesh is an important product trait of melons, and unique colors are often more popular with consumers. The color of melon pulp has rich variation types, mainly including orange red, light orange, green, white and the mixture of these colors (WatanabeK, SaitoT, HirotaS, TakahashiB, FujishitaN.1991. Carotenoidpigmentsinorange, lightorange, greenandwhitefleshcoloredfruitsofmelon(CucumismeloL.). Journal of the Japanese Society for Food Science and Technology, 38: 153–159.). The variety of pulp colors makes the breeding of melons develop in a diversified dire...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2537/161C12Q2563/107
Inventor 许勇张春秋宫国义张海英郭绍贵
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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