Molecular marker for identifying wild state/mutation state of Chinese cabbage TNL-E gene and applications of marker

A Chinese cabbage and labeling technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of R gene cloning that has not yet been seen, so as to avoid the blindness of selection, simplify screening methods, and genetically rich background effects

Inactive Publication Date: 2013-09-11
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
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  • Application Information

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  • Molecular marker for identifying wild state/mutation state of Chinese cabbage TNL-E gene and applications of marker
  • Molecular marker for identifying wild state/mutation state of Chinese cabbage TNL-E gene and applications of marker
  • Molecular marker for identifying wild state/mutation state of Chinese cabbage TNL-E gene and applications of marker

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1. Cloning of TNL-E in two resistant / susceptible Chinese cabbage inbred lines 8407 and 06-247

[0027] 1.1 Chinese cabbage genomic DNA extraction

[0028] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0029] (2) After the liquid nitrogen evaporates to dryness, transfer it to a 2ml centrifuge tube immediately, add about 0.6ml of CTAB extract preheated to 65°C for every 100mg of material, after melting, vigorously shake and mix the sample, place it in a 65°C water bath for 40- 60 minutes to lyse the cells;

[0030] (3) After the lysis is complete, take out the sample and let it cool down to room temperature completely. Add an equal volume of chloroform (chloroform), gently invert to mix, and place at room temperature for 10 minutes;

[0031] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0032] (5) Use a pipette to carefully suck out the upper aqueou...

Embodiment 2

[0053] Embodiment 2 Exploitation and Application of Overt ASM Markers

[0054] Since the primer combination TNL1F / TNL1R can amplify a 1367bp band in the disease-resistant material 8407, but no band can be amplified in the susceptible material 06-247, this pair of primers can be used to distinguish whether the TNL-E gene is complete Existing tags. Since the R gene is generally dominantly inherited, it can be detected when there is a wild-type gene in the genome; the mutant cannot be detected only in the homozygous state. The marker is 1367bp long, as shown in SEQ ID No.1, and the forward and reverse primer sequences are shown in SEQ ID No.3 and SEQ ID No.4. In order to verify the practicability of the marker, we used it to detect the backcross offspring of (8407×06-247×06-247. The specific implementation rules are as follows:

[0055] (1) The genomic DNA of each individual plant in the backcross population was extracted as described in 1.1 of Example 1.

[0056] (2) PCR ampl...

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Abstract

The invention discloses a dominant marker ASM-TNL-E-01 for identifying a wild state/mutation state of a Chinese cabbage TNL-E family gene, wherein a marker segment is 1367bp in size and a nucleotide sequence of the marker segment is as shown in SEQIDNo.1. Primers for identifying the specific molecular marker include a forward primer TNL1F:5'-TGAAACTTTCATTCAGCTTCAAGT-3' and a reverse primer TNL1R:5'-TTGGGTTAGTTTTCAACTTTCACA-3'; the sequences of the two primers are as shown in SEQIDNO.2 and SEQIDNO.3. The molecular marker can be used for identifying Chinese cabbage germ-plasm resources and assisting selective breeding, and a wild-type germ-plasm material or transformed offspring containing the TNL-E in 8407 is selected. A detailed method comprises the steps of: implementing PCR (Polymerase Chain Reaction) amplification on a genomic DNA (Deoxyribose Nucleic Acid) of an individual to be detected by utilizing the primers; detecting whether an amplified segment is generated; if a band is amplified, determining that a genome contains wild-type genes; otherwise, determining that the gene is mutated.

Description

technical field [0001] The present invention relates to the development and application of a gene mutant and its related site-specific molecular markers, in particular to the development and application of a specific molecular marker for distinguishing the wild type and mutant of the TNL-E family R gene in Chinese cabbage . The marker of the invention can be used to select materials containing wild-type resistance loci, and provides means of auxiliary selection for Chinese cabbage disease-resistant breeding, and belongs to the field of biotechnology. Background technique [0002] There are abundant disease resistance genes (R genes) in plant genomes, which ensure that they can resist a wide variety of pathogenic microorganisms throughout the growth and development cycle. More than 100 R genes have been cloned so far. In-depth analysis found that these R gene-encoded products generally contain leucine zippers (Leucine zippers, LZ), nucleic acid binding sites (Nucleotidebindi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 刘栓桃赵智中张晓燕张志刚李巧云王淑芬卢金东徐文玲刘贤娴付卫民崔莎莎田焕焕
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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