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Molecular marker for indicating and identifying character of orange pulp of melon, and primer and application thereof

A molecular marker and molecular marker-assisted technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of less use of CAPS molecular markers, so as to speed up the breeding work efficiency and shorten the breeding time. The effect of increasing the number of years and improving breeding efficiency

Inactive Publication Date: 2017-06-13
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, the main molecular markers used in melon breeding are RFLP, SSR, AFLP, RAPD, etc. CAPS molecular markers are rarely used

Method used

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  • Molecular marker for indicating and identifying character of orange pulp of melon, and primer and application thereof
  • Molecular marker for indicating and identifying character of orange pulp of melon, and primer and application thereof
  • Molecular marker for indicating and identifying character of orange pulp of melon, and primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Development of Molecular Markers and Primers

[0033] In this example, the orange melon line MR-1 and the green melon line X191 were selected as experimental materials to develop CAPS molecular markers. The MR-1 and X191 melon lines were sequenced by high-throughput sequencing technology, and the sequence of the coding region of the CmOr gene of the melon was obtained through the GenBank website. By comparing the sequence with the sequencing material, the SNP mutation in the coding region of the CmOr gene of the melon was obtained. Interval, within this interval, the base segment with SNP mutation site in the sequencing data is mined, the enzyme digestion information of the SNP site is analyzed, and the sequence with CAPS mutation is obtained, that is, the CAPS molecular marker HF.

[0034] The melon CmOr gene sequence is located between chromosome 9 5207122-5214439. The nucleotide sequence of the molecular marker HF is shown in SEQ ID NO.1, wherein the 640t...

Embodiment 2

[0039] Embodiment 2: extraction of genomic DNA and PCR reaction

[0040] The leaves of MR-1 and X191 melon populations were collected, and the genomic DNA was extracted by the improved CTAB method.

[0041] The obtained CAPS primers and genomic DNA were used for PCR reaction, and the PCR reaction system was shown in Table 2 (20 μL):

[0042] Table 2 PCR reaction system

[0043]

[0044] Amplification was carried out by touchdown PCR (TD-PCR), the program was: 94°C pre-denaturation for 7 min, 94°C denaturation for 1 min, 60°C annealing for 30 s, 0.5°C reduction per cycle, 72°C extension for 90 s, 30 cycles; 94 Denaturation at ℃ for 30s, annealing at 45℃ for 30s, extension at 72℃ for 30s, 10 cycles; extension at 72℃ for 10min, storage at 4℃.

Embodiment 3

[0045] Example 3: Enzyme digestion verification of PCR products

[0046] Carry out the enzyme digestion reaction according to the Hinf I restriction endonuclease operation guide, and perform enzyme digestion verification on the obtained PCR product. The enzyme digestion system is shown in Table 3: (15.3 μL)

[0047] Table 3 enzyme digestion reaction system

[0048]

[0049]

[0050] Enzyme digestion in 37°C water bath for 3-4 hours, and the digested products were detected by 1% agarose electrophoresis, and the detection results were as follows: figure 1 shown. X191 (green) was not cleaved by restriction endonucleases due to the absence of enzyme cleavage sites, and was still 946bp. Due to the presence of enzyme cleavage sites in MR-1 (orange), it was cleaved by HinfⅠ and obtained 640bp and 306bp fragment. The result is as figure 1 shown.

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Abstract

The invention discloses a molecular marker for indicating and identifying the character of an orange pulp of a melon, and a primer and application thereof and belongs to the technical field of molecular marker assisted selection breeding of the melon. The molecular marker HF is shown as SEQ ID NO.1, wherein the melon is identified as the melon with the orange pulp if the No.640 locus basic group m of the nucleotide sequence shown as SEQ ID NO.1 is G. The invention also provides the primer of the molecular marker HF and a method for utilizing the primer to identify the character of the orange pulp of the melon. According to the identifying method, the assistant selection can be performed on the color of the melon pulp in the seedling stage of the melon; the operation is simple and convenient, and the result is clear; the breeding efficiency is greatly increased, and the breeding cycle is shortened; and the breeding efficiency is increased. The molecular marker HF, the primer pair thereof and the identifying method can be used for molecular marker assistant breeding for the melon, assistant identifying and assistant breeding for the character of the pulp of the melon and screening for the character of the pulp of the melon.

Description

technical field [0001] The invention relates to a molecular marker for predicting and identifying the character of orange pulp of melon, its primer and application, and belongs to the technical field of molecular marker-assisted selection breeding of melon. Background technique [0002] Molecular marker assisted selection (MAS) is a new technology produced with the rapid development of modern molecular biology technology. Direct selection, molecular breeding. Molecular marker-assisted selection breeding is a modern molecular breeding technique that uses DNA markers or functional genes linked to important crop traits to improve plant varieties. Therefore, the key to the success of molecular marker-assisted selection lies in whether the obtained molecular markers are consistent with the traits to be analyzed Linkage and whether there is commonality between different materials of the same species. [0003] The goal of melon breeding involves the improvement of certain economi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 高鹏赵玉龙栾非时刘识王学征朱子成
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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