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Method for positioning and cloning double-recessive gene for controlling same trait in plant

A technology of recessive genes and traits, applied in the field of positioning and cloning double recessive genes that control the same trait in plants, can solve the problems of long time spent

Active Publication Date: 2018-09-28
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has high precision and is often used for fine mapping and map-based cloning of genes; however, it takes a long time because the positioning is usually carried out in the high generation of backcross

Method used

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  • Method for positioning and cloning double-recessive gene for controlling same trait in plant
  • Method for positioning and cloning double-recessive gene for controlling same trait in plant
  • Method for positioning and cloning double-recessive gene for controlling same trait in plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the positioning and map-based cloning of genes ws1a and ws1b

[0044] 1. Initial mapping of genes ws1a and ws1b

[0045]1. A chlorophyll-deficient white stem mutant white stem1 (ws1) was screened from the common tobacco variety Zhongyan 100 (ZY) induced by EMS. The method of allele test (see the following literature for the specific method: Mapping of twowhite stem genes in tetraploid common tobacco (Nicotiana tabacco L.)) was used for genetic analysis, and the specific steps were as follows: the white stem mutant ws1 was crossed with the Burley tobacco variety, The F1 generation is obtained, and the performance of the F1 generation is the same as that of the parents; then the F1 generation is self-crossed to obtain self-crossed offspring, and the traits of the self-crossed offspring do not segregate. Genetic analysis showed that the white stem trait was controlled by two recessive nuclear genes ws1a and ws1b, and was allelic with the white stem gene of B...

Embodiment 2

[0056] Embodiment 2, application of metalloprotease WS1A or WS1B in regulating plant chloroplast metabolism

[0057] 1. Obtaining WS1A Tobacco and WS1B Tobacco

[0058] 1. Construction of recombinant vector

[0059] (1) Cloning of genes WS1A and WS1B

[0060] (1-1) RNA was extracted from leaves of Dajinyuan safflower, and cDNA was reverse transcribed. RNA extraction and reverse transcription were completed using MiniBEST plant RNA Extraction Kit (Code No.9769) from TaKaRa Company and PrimerScript II 1st Strand cDNA synthesis Kit (Code No.6210) from TaKaRa Company, respectively. The cDNA was used as a template and primers CW-1F / CW-1R were used to amplify to obtain PCR products.

[0061] (1-2) WS1A and WS1B were identified respectively after PCR products were cloned by TA (Mighty TA-cloning Kit (TaKaRa Company, Code No.6028)) and sequenced. The WS1A gene sequence is shown in sequence 3, and the WS1B gene sequence is shown in sequence 4.

[0062] (2) Construction of expressi...

Embodiment 3

[0092] Example 3 Molecular markers co-segregated with Burley tobacco control genes

[0093] 1. Primer design for WS1A, ws1a, WS1B and ws1b genotype identification

[0094] 1. Download the full gene sequence of WS1A and WS1B from the Honghua Dajinyuan Genome Database, and use the online analysis tool MUSCLE (https: / / www.ebi.ac.uk / Tools / msa / muscle / ) for sequence comparison to obtain WS1A For the sequence difference between WS1B and WS1B, intercept the genome sequence of 200-400bp upstream and downstream of ws1a and ws1b mutation sites for future use;

[0095] 2. Since the WS1A and WS1B sequences are highly homologous, use the sequence differences between the two to design specific PCR primers containing gene mutation sites:

[0096] (1) For WS1A and ws1a, a pair of specific primers 1325-2F / 1325-1R (namely M-a) was designed based on the 8-base length difference between the two, and the PCR products were separated by polyacrylamide gel electrophoresis. Those that are similar to ...

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Abstract

The invention discloses a method for positioning and cloning a double-recessive gene for controlling the same trait in a plant. According to the method, a positioning group of which a target trait isseparated can be quickly constructed starting from a common conventional material. Meanwhile, an unconventional material such as a DH system is not needed, and the gene can be subjected to careful positioning and map-based cloning at the BC1F1 generation. The method has the advantages of being simple, quick and efficient, and capable of being widely applied to conduct careful positioning and map-based cloning on the double-recessive gene for controlling the same trait.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for locating and cloning double recessive genes controlling the same character in plants. Background technique [0002] Double recessive genes controlling the same trait are mostly produced by genome doubling or gene duplication, and are most common in allotetraploid plants, such as common tobacco, Brassica napus, durum wheat, etc., and occasionally in diploid plants, such as rice , corn, etc. Due to the completely redundant biological functions of such genes, the mutation of a single gene cannot lead to the corresponding recessive phenotype. [0003] Molecular markers are currently the most important and reliable means of gene mapping. Regarding the use of molecular markers to locate double recessive genes controlling the same trait, there are currently two methods according to the segregation population used: one is to use F2 or BC1F1 The populations were separated, and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895A01H1/02
CPCA01H1/02C12Q1/6895C12Q2600/156
Inventor 吴新儒龚达平刘贯山王大伟陈梦
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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