Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer

A functional gene and gene technology, applied in the field of molecular biology, can solve the problems of low detection efficiency, long time, difficulty in meeting production needs, etc.

Active Publication Date: 2015-12-02
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although these functional markers have been partially applied in the detection of wheat functional genes, the operation process is cumbersome and complicated, requiring enzyme digestion and electrophoresis, and the detection efficiency is low.
In the breeding process, breeders screen a large number of progeny materials, which requires high cost and long time, and this method is difficult to meet production needs

Method used

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  • Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer
  • Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer
  • Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. Design of KASP markers for functional genes of important traits in wheat and development of special primer sequences

[0094] Wheat functional genes involved in the present invention include Ppo-D1 gene, TaPod-A1 gene, TaCKX6-D1 gene, TaCW1-4A gene, TaGS-D1 gene, TaGASR7-A1 gene, 1-FEHw3 gene, Pinb2-v2 gene, Psy- B1 gene, Psy1-D1 gene, TaZds-A1 gene, Talyce-B1 gene, TaPds-B1 gene, Glu-B1 gene.

[0095] 1. Acquisition of allelic variant sequences of wheat functional genes

[0096] Enter the above-mentioned wheat functional gene names TaGS-D1 and TaZds-A1 in the NCBI database Nucelotide to obtain two variant forms of the functional gene in wheat and download the sequence. Ppo-D1 gene, TaPod-A1 gene, TaCKX6-D1 gene, TaCW1-4A gene, TaGASR7-A1 gene, 1-FEHw3 gene, Pinb2-v2 gene, Psy-B1 gene, Psy1-D1 gene, Talyce -variant sequences of B1 gene, TaPds-B1 gene, Glu-B1 gene.

[0097] The sequence and functional markers of the Ppo-D1 gene were published in the documen...

Embodiment 2

[0128] Example 2, Establishment of KASP Marker Detection System for Wheat Functional Genes

[0129] 1. Genomic DNA extraction

[0130] The leaf tissue of the wheat variety to be tested was taken, and the whole genome DNA of the leaf was extracted by CTAB method.

[0131] 2. Using the genomic DNA extracted in step 1 as a template, use the special primers developed in Example 1 for detecting the KASP markers of the 14 functional genes to perform PCR amplification respectively to obtain PCR amplification products.

[0132] Preparation of KASP-labeled primer working solution:

[0133] Take 12 μl of upstream primers (100 μM) and 30 μl of downstream primers (100 μM), supplement them with sterile ultrapure water to 100 μl, and use them as KASP-labeled primer working solutions for use.

[0134] Reaction system for PCR amplification: template DNA 1.76μl (concentration: 30-50ng / μl), primer working solution 0.045μl, 50mM MgCl 2 (LGC Company, Lot No. 10364672), KASP2×MasterMix 2 μl (LG...

Embodiment 3

[0157] Embodiment 3, the application of the method for detecting wheat functional genes with KASP markers in breeding

[0158] 1. Adopt the embodiment of the present invention 1 to develop KASP markers to detect the genotypes of 14 functional genes of 121 wheat varieties (see Table 4) planted in the Huanghuai wheat region of Anyang, Henan Province. Refer to Example 2 for specific operations, and obtain the genotypes of each variety. Genotypes of functional genes, see Table 4 for specific results.

[0159] Genotyping diagram of some wheat samples Figure 1-Figure 14 As shown, the sample shown in black in the lower left corner is the blank control of each PCR plate, and the genotype of the blue sample aggregated near the X axis is the allele type connected to the FAM fluorescent tag sequence, aggregated near the Y axis The genotype of the sample shown in red is the allelic type connected to the HEX fluorescent label sequence, the genotype of the sample shown in green in the mid...

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Abstract

The invention discloses a primer set for detecting functional genes of wheat on the basis of a KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of the set primer. The primer set comprises totally 14 groups of KASP primers. The KASP primers are respectively designed for the 14 functional genes of the wheat, and particular nucleotide sequences of the KASP primers are sequences 1-42 in sequence tables. The primer set and the application have the advantages that the functional genes of different wheat varieties can be quickly detected by the aid of the KASP primer set, methods for detecting the functional genes of the wheat are simple, convenient and speedy, and detection results are accurate and reliable; the primer set has an important theoretical significance and important economic value in assistant selection of the wheat varieties by the aid of molecular markers.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a set of primers for detecting wheat functional genes based on KASP technology and an application thereof. Background technique [0002] In recent years, molecular markers based on DNA sequence polymorphisms have been highly valued by domestic and foreign breeders. The use of molecular markers closely linked to target traits for selection is not affected by the environment, and the selection results are reliable. In addition, when aggregating favorable traits, genetic background selection can be used in the backcross infiltration process to reduce linkage burden and accelerate the breeding process. Therefore, the development of molecular markers to control important traits of wheat by means of modern molecular biology is of great significance to wheat breeding in my country. [0003] So far, molecular markers commonly used in wheat genetics and breeding include RFLP, RAPD, AFLP, S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 何中虎阿韦斯·拉希德卢家玲夏先春
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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