Ma pomelo InDel (insertion-deletion) molecular marker, and application of molecular marker in differentiating rough bark Ma pomelo at early stage of citrus seed seedling

A molecular marker, Majia pomelo technology, applied in the field of genetics and breeding, can solve problems such as easy mixing, impure seedlings, loss, etc., and achieve the effects of high accuracy, reducing breeding workload and eliminating interference.

Active Publication Date: 2018-10-16
HUAZHONG AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0003] However, with the development of the Majia pomelo industry and the deepening of investigation and research, it was found that the morphological properties of Majia pomelo are very different and not stable, and people divide Majia pomelo into rough peel fruit (high acid type) and There are two types of fine-skinned fruit (medium acidity). The thin-skinned fruit is better than the rough-skinned fruit in terms of flavor and taste, but it is difficult to distinguish morphologically from the appearance of the seedlings, especially in the current breeding process of Majia pomelo seedlings. The types of fine skin are often mixed, and seedling factories have no way to distinguish them, which brings risks to the Majia pomelo industry
Majia pomelo usually starts to bear fruit after 4-5 years. If the seedlings are found to be impure after 5 years, it has caused great losses and brought difficulties to orchard management.

Method used

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  • Ma pomelo InDel (insertion-deletion) molecular marker, and application of molecular marker in differentiating rough bark Ma pomelo at early stage of citrus seed seedling
  • Ma pomelo InDel (insertion-deletion) molecular marker, and application of molecular marker in differentiating rough bark Ma pomelo at early stage of citrus seed seedling
  • Ma pomelo InDel (insertion-deletion) molecular marker, and application of molecular marker in differentiating rough bark Ma pomelo at early stage of citrus seed seedling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] (1) Genomic DNA of fine-skinned Majia pomelo (you series), rough-skinned Majia pomelo, Guanxi honey pomelo, Shatian pomelo and local soil pomelo in Guangfeng County, Jiangxi Province was extracted and prepared by the improved CTAB method.

[0058] (2) Using the primer pair chr2.10-9584F / chr2.10-9584R to amplify and analyze the genomic DNA of all the citrus above. The amplification reaction was carried out in a PTC-200 PCR machine (MJ. Research Inc., USA). The reaction system for PCR amplification is 7μl of Mix solution, 0.3μl of 10mmol / L forward primer, 0.3μl of 10mmol / L reverse primer, 1.5μl of 100ng / ul template DNA and 6μl of ddH 2 O. The specific steps are: denaturation at 95°C for 5 minutes; followed by 35 cycles: 94°C, 30s, 55°C, 30s, 72°C, 35s; finally 72°C, 10min, 12°C, 30min, and 4°C storage.

[0059] (3) The amplified product was detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank, using 1×TAE buffer (0.04M Tris-acetate, 0.001M E...

Embodiment 2

[0062] (1) Genomic DNA of Majia pomelo and Guanxi pomelo were extracted and prepared by the improved CTAB method.

[0063] (2) Using the primer pair p-chr6.21-8689F / p-chr6.21-8689R to amplify and analyze the genomic DNA of all the above citrus. The amplification reaction was carried out in a PTC-200 PCR machine (MJ. Research Inc., USA). The reaction system for PCR amplification is 7μl of Mix solution, 0.3μl of 10mmol / L forward primer, 0.3μl of 10mmol / L reverse primer, 1.5μl of 100ng / ul template DNA and 6μl of ddH 2 O. The specific steps are: 95°C denaturation for 5 minutes; followed by 35 cycles: 94°C, 30s, 55°C, 30s, 72°C, 35s; finally 72°C, 10min, 12°C, 30min, 4°C storage.

[0064] (3) The amplified product was detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank, using 1×TAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 120V, electrophoresis for 20min. After electrophoresis, the gel imaging system (UVP) took pictures and saved them...

Embodiment 3

[0067] (1) 80 random seedling plants of Majia pomelo (excellent line) mixed with rough skin Majia pomelo were used as test materials, and the genomic DNA of the above test materials was extracted and prepared by the improved CTAB method.

[0068] (2) Using the primer pair p-chr6.21-8689F / p-chr6.21-8689R to amplify and analyze the genomic DNA of all the above citrus. Amplification reactions were performed in a PTC-200 PCR instrument (MJ. Research Inc., USA). The reaction system for PCR amplification is 7μl of Mix solution, 0.3μl of 10mmol / L forward primer, 0.3μl of 10mmol / L reverse primer, 1.5μl of 100ng / ul template DNA and 6μl of ddH 2 O. The specific steps are: 95°C denaturation for 5 minutes; followed by 35 cycles: 94°C, 30s, 55°C, 30s, 72°C, 35s; finally 72°C, 10min, 12°C, 30min, 4°C storage.

[0069] (3) The amplified product was detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank, using 1×TAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0),...

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Abstract

The invention provides a Ma pomelo InDel (insertion-deletion) molecular marker, and an application of the molecular marker in differentiating a rough bark Ma pomelo at an early stage of a citrus seedseedling, and belongs to the field of genetic seed breeding. The Ma pomelo InDel molecular marker comprises a fragment obtained by amplification of a primer pair chr2.10-9584F / chr2.10-9584R, and a fragment obtained by amplification of a primer pair p-chr6.21-8689F / p-chr6.21-8689R. The molecular marker provides a novel molecular tool for identification and breeding of the genetically stable Ma pomelo seed seedling and early screening. The InDel molecular marker can be used for differentiating the rough bark Ma pomelo in a citrus variety at the early stage of the seed seedling, and has importantsignificance for identification of the citrus variety and risk control in Ma pomelo production.

Description

technical field [0001] The invention belongs to the field of genetic breeding, and in particular relates to a Majiayou InDel molecular marker and its application in distinguishing rough-skinned Majiayou at the early stage of citrus seedlings. Background technique [0002] Pomelo (Citrus grandis) is a citrus fruit of Rutaceae, which has a long history of cultivation in my country. Pomelo is an auspicious fruit in Chinese traditional culture, and is often used in weddings, sacrifices and other activities. Residents in the south of our country like to plant pomelo in front of their houses. Most of these cases are propagated by seeds, and a wealth of local varieties and strains have been formed during the long-term cultivation process. However, the genetic background and origin of these grapefruits are not clear at present, and there are often cases of the same thing with different names or the same name with different things. Guangfeng County, Jiangxi Province and its surroun...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 徐强万鹏飞王沦罗鑫路志浩蒋小林方秋莹邓秀新
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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