Citrus mitochondrion InDel molecular markers and application thereof

A molecular marker, mitochondrial technology, applied in recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problems of poor specificity and narrow application range of citrus, achieve strong stability, save identification time, distinguish high rate effect

Active Publication Date: 2020-01-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the mitochondrial molecular markers currently used in citrus are developed based on the mitochondrial genomes of crops such as Arabidopsis thaliana and rapeseed, and their application range in citrus is narrow and their specificity is poor.

Method used

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  • Citrus mitochondrion InDel molecular markers and application thereof
  • Citrus mitochondrion InDel molecular markers and application thereof
  • Citrus mitochondrion InDel molecular markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] (1) DNA extraction: Genomic DNA of 62 varieties of Citrus, Hovenia citrus, and kumquat were extracted by CTAB method, and the variety numbers are shown in figure 1 Description of drawings.

[0059] (2) PCR amplification: Utilize primer pair HBF / HBR, G1F / G1R, ZKF / ZKR, SJGF / SJGR to carry out PCR amplification to the genomic DNA of above-mentioned 62 varieties respectively, utilize primer pair BTCF / BTCR pair No. 1-25 varieties were amplified by PCR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C.

[0060] (3) Electrophoresis detection: use 2.5% agarose gel electrophoresis to detect the amplified product, the conditions of the electrophoresis solution are: 1×TAE buffer solution (0.04M Tris-...

Embodiment 2

[0063] (1) DNA extraction: Genomic DNA of cytoplasmic hybrids Huayou 2, Guoqing 1 Wenzhou mandarin and Huayou 1 were extracted by CTAB method.

[0064] (2) PCR amplification: the genomic DNA of the above-mentioned citrus varieties was amplified by using the primer pair HBF / HBR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C.

[0065] (3) Electrophoresis detection: use 2.5% agarose gel electrophoresis to detect the amplified product, and the conditions of the electrophoresis solution are: 1×TAE buffer solution (0.04M Tris-acetate, 0.001M EDTA, pH=8.0), voltage 60V , electrophoresis 80min. After electrophoresis, use the gel imaging system (UVP) to take pictures of the gel and save the pictures. ...

Embodiment 3

[0068] (1) DNA extraction: Genomic DNA of cocktail grapefruit, Shatian pomelo, National Day No. 1 Wenzhou mandarin and rock sugar orange were extracted by CTAB method.

[0069] (2) PCR amplification and electrophoresis detection 1: Genomic DNA of cocktail grapefruit, Shatian pomelo, rock sugar orange, and National Day No. 1 Wenzhou mandarin were amplified by using the primer pair HBF / HBR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C. The amplified product was detected by 2.5% agarose gel electrophoresis, 1×TAE buffer solution (0.04M Tris-acetate, 0.001M EDTA, pH=8.0), voltage 60V, electrophoresis for 80min. After electrophoresis, use the gel imaging system (UVP) to take pictures of the gel an...

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Abstract

The invention discloses a set of citrus mitochondrion InDel molecular markers and application thereof, and belongs to the technical field of plant molecular marker assisted germplasm resource identification. Primer sequences of the 5 pairs of the citrus mitochondrion InDel molecular markers are shown as SEQ ID NO.1-10. Germplasms of citrus, poncirus and fortunella hindsii swingle can be rapidly and accurately identified by using primers of the citrus mitochondrion InDel molecular markers, the mitochondrial origin of a citrus protoplast fusion progeny can be identified, and the female parent origin of parent-unknown citrus cultivars can be analyzed. The citrus mitochondrion InDel molecular markers are of great significance in protecting and utilizing wild citrus resources.

Description

technical field [0001] The invention belongs to the technical field of identification of germplasm resources assisted by plant molecular markers, in particular to a group of mitochondrial InDel molecular markers for identifying germplasm resources of citrus, mandarin orange and trifoliate genus and application thereof. Background technique [0002] Citrus is a citrus plant of Rutaceae (Rutaceae), which originated in the Himalayas of my country. It is the largest fruit tree in the world and the most important fruit tree in southern my country. There are many kinds of citrus and its close relatives. Citrus has the characteristics of apomixis and frequent clonal variation, and different species are easy to hybridize. For example, plants such as Citrus and Citrus can hybridize with each other. Hybridization is frequent, so the nuclear genetic background of citrus plants is more complex, and the specificity of nuclear molecular marker primers is poor. At the same time, as a matu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 郭文武李超超张帅解凯东伍小萌徐强邓秀新
Owner HUAZHONG AGRI UNIV
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