Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof

An auxiliary identification and drought resistance technology, applied in the direction of biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems affecting wheat yield, etc.

Active Publication Date: 2021-11-16
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the global wheat planting area and yield have increased by leaps and bounds, abiotic adversities such as drought, high tempera

Method used

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  • Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof
  • Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof
  • Method for auxiliary identification of drought resistance of wheat to be detected and special molecular marker thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Cloning of embodiment 1, TaPYL1 gene

[0072] 1. Take the seeds of Chinese spring, accelerate germination at 25°C for 3 days, then transfer the germinated seeds to nutrient soil, and cultivate them at 25°C for two weeks to obtain Chinese spring seedlings.

[0073] 2. Extract the total RNA of Chinese spring seedlings, and then reverse transcribe to obtain the cDNA of Chinese spring.

[0074] 3. Using the cDNA of Chinese Spring as a template, PCR amplification was performed using a primer pair consisting of 5'-ATGGAGCAGCAGCCTGTG-3' (SEQ ID NO: 3) and 5'-TTATTCTGCCGGCGGCGC-3' (SEQ ID NO: 4) to obtain A PCR amplification product of about 642bp.

[0075] 4. Sequencing the PCR amplification products.

[0076] Sequencing results showed that the nucleotide sequence of the PCR amplification product is shown in SEQ ID NO:2.

[0077] The gene shown in SEQ ID NO: 2 was named TaPYL1 gene. The TaPYL1 gene encodes a TaPYL1 protein, and the amino acid sequence of the TaPYL1 protein...

Embodiment 2

[0078] Embodiment 2, acquisition and drought resistance identification of transgenic TaPYL1 wheat

[0079] The solute and its concentration of 10×LS Major is CaCl 2 2H 2 O44g / L, KH 2 PO 4 1.7g / L, KNO 3 19g / L, MgSO 4 ·7H 2 O37g / L and NH 4 NO 3 16.5g / L, the solvent is water.

[0080] 100×LS Minor's solute and its concentration as CoC l2 ·6H 2 O2.5mg / L, CuSO 4 ·5H 2 O2.5mg / L, H 3 BO 3 620mg / L, KI83mg / L, MnSO 4 ·5H 2 O2230mg / L and ZnSO 4 ·7H 2 O1060mg / L, the solvent is water.

[0081] The solute of 100×Fe-EDTA and its concentration is FeSO 4 ·7H 2 O2.78g / L and Na 2 EDTA3.73g / L, the solvent is water.

[0082] The solute and its concentration of 100×Vitamin are Pyridoxine-HCl 0.05g / L, Nicotinic acid0.05g / L, Thiamine-HCl 0.1g / L and myo-Inositol 10g / L, and the solvent is water.

[0083] Selection medium: 100mL10×LS Major, 10mL100×LS Minor, 10mL100×Fe-EDTA, 10mL100×Vitamin, 5mL2,4-D, 40gMaitose, 0.5g Glutamine, 0.75g MgCl 2 ·6H 2 O, 1.95g MES and 5g Agarose wer...

Embodiment 3

[0132] Example 3. Haplotypes related to drought resistance of wheat and their molecular markers and applications

[0133] 1. Development of Molecular Markers

[0134] 1. After a large number of experiments, the inventors of the present invention found that there are two haplotypes related to drought resistance in the polymorphic site upstream of the initiation codon ATG of the TaPYL1 gene in the wheat genomic DNA, named respectively as haplotype A and haplotype B, such as Figure 5 shown.

[0135] 2. Preparation of specific primer pairs for identifying molecular markers related to drought resistance in wheat. The specific primer pair consisted of primer F: -CGAAGAATTGGTGAATCATGTACTAC-3' (SEQ ID NO: 12) and primer R: 5'-TAAAAAATAGAAGAGCATCTCCTAAAAG-3' (SEQ ID NO: 13).

[0136] 2. Polymorphism detection

[0137] 1. The drought-resistant wheat variety Pubing202 was used as the male parent, and the drought-sensitive wheat variety GLUYAS EARLY was used as the female parent to cro...

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Abstract

The invention discloses a method for auxiliary identification of drought resistance of wheat to be detected and a special molecular marker thereof. The method comprises the following steps: by taking genome DNA of wheat to be detected as a template, carrying out PCR amplification by adopting a primer pair consisting of a primer F and a primer R to obtain a DNA fragment; the DNA fragment being the molecular marker related to wheat drought resistance. The molecular marker specifically can be a DNA segment A or a DNA segment B. The nucleotide sequences of the primer F, the primer R, the DNA segment A and the DNA segment B are shown as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 in sequence. The wheat variety of which the genome contains the DNA segment B and does not contain the DNA segment A has drought resistance. By applying the molecular marker provided by the invention, the wheat variety with drought resistance can be quickly screened out, so that the breeding pace of the wheat variety is accelerated. The method has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for assisting in identifying the drought resistance of wheat to be tested and a special molecular marker thereof. Background technique [0002] Plants grow and develop in a complex and changeable environment, and are often subjected to adversity stress, among which drought is the main stress factor that affects and limits plant growth and development, and even causes plant death, seriously affecting agricultural production. Therefore, cultivating drought-resistant crop varieties has always been one of the main goals of agricultural science and technology research. [0003] Wheat (Triticum maestivum L.) is the most important carbohydrate and protein source for people all over the world. Although the global wheat planting area and yield have increased by leaps and bounds, abiotic adversities such as drought, high temperature, and high salinity have seriously affec...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 毛虎德康振生简超黄雪玲程新秀
Owner NORTHWEST A & F UNIV
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