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Banana vascular wilt resistant molecular marker and application thereof

A banana fusarium wilt, molecular marker technology, applied in the direction of DNA/RNA fragment, microbial determination/inspection, recombinant DNA technology, etc. Simple and stable effect

Active Publication Date: 2018-02-02
东莞市香蕉蔬菜研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The disease resistance mechanism of banana Fusarium wilt is relatively complicated, and there are many genes related to disease resistance. At present, there are still relatively few molecular methods that can be directly and effectively used in banana breeding. The identification method of Fusarium wilt banana, the research in this area is of great significance to the disease-resistant breeding of banana

Method used

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  • Banana vascular wilt resistant molecular marker and application thereof
  • Banana vascular wilt resistant molecular marker and application thereof
  • Banana vascular wilt resistant molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The DNA extraction of embodiment 1 banana leaf

[0037] Banana (genotype AAA) varieties (Table 1) with different resistance to banana wilt were selected for DNA extraction.

[0038] The DNA extraction method adopts the modified CTAB method, and the specific operation is as follows:

[0039] (1) extract the DNA in the banana leaf, choose the young leaf that does not have disease and insect damage newly, rinse with water, get 0.2g after drying, grind with liquid nitrogen, add a small amount of cross-linked polyvinylpyrrolidone (PVPP) in the process of grinding ), quickly grind the leaves to powder.

[0040] (2) Add 800 μl of preheated 4×CTAB to the ground sample, mix well, bathe in water at 65° C. for 30 minutes, shake the sample 3 times during this period, and centrifuge at 12,000 rpm for 10 minutes.

[0041] (3) Add 600 μl of chloroform-isoamyl alcohol (24:1), mix well, and centrifuge at 12000 rpm for 6 min.

[0042] (4) Aspirate the supernatant, repeat step 3, and p...

Embodiment 2

[0051] Example 2 Banana Fusarium Wilt Resistance SRAP Marking and Detection

[0052] Ten banana varieties were selected, namely Nongke No.1, Nantianhuang, Dongjiao No.1, BMX51, Kangku No.1, G30, Williams, Dongguan Zhongba, Brazil, and L3-3. Using SRAP primers me9 / em5, PCR amplification was carried out using the DNA of these 10 banana varieties as templates, and the amplified products were detected.

[0053] (1) SRAP Marker for Banana Fusarium Wilt

[0054] 10 banana varieties were amplified by PCR using SRAP primers me9 / em5, and the amplified products were detected by gel electrophoresis, observed and photographed in a gel imager. The result is as figure 1 As shown, M: 2000bp marker: 1-10 samples: Nongke 1, Nantianhuang, Dongjiao 1, BMX51, Kangku 1, G30, Williams, Dongguan Zhongba, Brazil, L3-3. Among them, five susceptible varieties, G30, Williams, Dongguan Zhongba, Brazil, and L3-3, produced specific bands of about 400 bp.

[0055] The SRAP primer sequence adopted is: me...

Embodiment 3

[0066] Example 3 Banana Fusarium Wilt Resistance SCAR Molecular Marker Specific Primer Design and Identification Method

[0067] Primers were designed using the primer online design tool NCBI / primer-BLAST, and a total of 8 pairs of primers were designed (see Table 4 for primer sequences). The seven varieties of Dongguan Zhongba and L1N2 were screened for primers and optimized conditions.

[0068] Table 4 SCAR primer sequence list

[0069]

[0070] The result is as image 3 As shown, M: 2000bp marker: Samples 1-7 are: Nongke 1, Nantianhuang, Dongjiao 1, Kangku 1, G30, Dongguan Zhongba, L1N2. After optimization of the PCR program and PCR system, the primer SCA11 / SCA12 amplifies the band length of 296 bp, and the 5th bp to the 300th bp of the specific band are amplified from the SRAP primer me9 / em5. The target band of the amplification product of primer SCA11 / SCA12 is clear and the result is accurate. Among them, the amplified products of susceptible varieties G30, Donggua...

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Abstract

The invention discloses an SCAR molecular marker for identifying banana vascular wilt. The molecular marker has the nucleotide sequence shown as SEQ ID:4. The invention also discloses a primer used for amplifying the SCAR molecular marker, a kit containing the primer and application of the molecular marker, the primer and the kit to identification of banana vascular wilt resistance or screening ofbanana with vascular wilt. The invention also discloses a method for identifying the banana vascular wilt resistance. A banana leaf DNA is used as a template, the primer is used for PCR amplificationreaction; amplification products are detected; the detection of whether a 296bp specific strip exists or not is used as the concrete expression. The SCAR molecular marker provided by the invention provides a molecular assistance technology for the banana disease-resistance new variety breeding; the SCAR molecular marker can be further used for the auxiliary screening of the banana new variety (line) using banana tissue culturing seedlings, banana field variant strains, physicochemical mutagenesis later generations and the like as screening groups.

Description

technical field [0001] The invention relates to a banana fusarium wilt resistance molecular marker, in particular to a SCAR molecular marker for banana fusarium wilt resistance identification, a molecular marker primer, a kit, an application and a method. Background technique [0002] Banana is an important fruit in the world, and there are 130 countries in the world that cultivate bananas. Banana export is the main means of earning foreign exchange in tropical and subtropical regions, and occupies a considerable proportion in its gross national product. my country is one of the main producing countries of bananas, and the banana industry occupies a very important position in the agricultural output value of my country's subtropical regions. [0003] Banana (Musa AAA Cavendish subgroup cv.) is the largest planted variety in the world, and it is also the main type of banana planted in my country. But Banana is seriously damaged by race 4 of banana wilt, even destructive dam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 王芳夏玲吕顺胡贝曾莉莎刘建平
Owner 东莞市香蕉蔬菜研究所
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