High-throughput detection markers of wheat powdery mildew resistance gene Pm5e and application of detection markers in breeding

A technology for powdery mildew resistance gene and wheat powdery mildew, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low polymorphism level, low detection efficiency, long genetic distance, etc. Achieve the effect of improving powdery mildew resistance, reducing impact and improving breeding efficiency

Active Publication Date: 2018-07-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

The predecessors used SSR markers to locate the gene, and initially located it on the 7BL chromosome, but the genetic distances between the two SSR markers and the target gene were 11cM and 6.6cM respectively (Huang et al. 2003), Wang Honggang et al. (2008) here Based on this, two SSR markers were developed, and the genetic distances were 5.1cM and 4.9cM respectively. Although these two SSR markers shortened the genetic distance between the Pm5e gene and the marker to a certain e

Method used

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  • High-throughput detection markers of wheat powdery mildew resistance gene Pm5e and application of detection markers in breeding
  • High-throughput detection markers of wheat powdery mildew resistance gene Pm5e and application of detection markers in breeding
  • High-throughput detection markers of wheat powdery mildew resistance gene Pm5e and application of detection markers in breeding

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1: Wheat genome DNA extraction

[0057] 1) Collection of leaves for DNA extraction

[0058] At the three-leaf stage of wheat, young leaves of wheat were selected and put into the corresponding 96-well deep-well plate according to the number. In order to prevent DNA degradation, the operation was performed on an ice box;

[0059] 2) Genomic DNA of wheat was extracted by CTAB method; stored at -20°C for future use.

[0060] The above steps are as follows:

[0061] (a) take young wheat leaves and place them in a 96-well deep-well plate, freeze them with liquid nitrogen and grind them into powder on a tissue grinder; (b) preheat them in each well of the 96-well deep-well plate Put 600 μL of CTAB extract at 65°C in a water bath at 65°C for 60 minutes, and shake gently every 10 minutes to fully lyse the DNA; (c) add 600 μL of chloroform-isoamyl alcohol mixture with a volume ratio of 24:1, and shake gently for 10 minutes ; (d) Centrifuge at 12000r for 10min at 4°...

Embodiment 2

[0077] Example 2: Development of Tightly Linked Markers in Pm5e Gene in Rejuvenation 30

[0078]Currently, 6 markers closely linked to Pm5e have been published, including 4 SSR markers (Xgwm783, Xgwm1267, Xwmc364 and Xbarc065) and 2 EST markers (CJ729392 and CJ584170). Due to the low specificity of SSR markers, the KASP detection marker developed in this study is based on two EST markers. The disease-resistant material Rejuvenation 30 contains the Pm5e gene, and Chancellor is highly susceptible to powdery mildew. Using these two resistant parents to create F 2 A total of 214 plants were isolated from the population, and the powdery mildew phenotype was identified at the seedling and adult plant stages. According to the identification results, select F with high resistance and high sensitivity respectively. 2 15 individual plants were used to construct resistance and susceptibility gene pools. Using 35K gene chip technology to identify the SNP sites of the anti-susceptibili...

Embodiment 3

[0079] Example 3: Primer dilution and KASP assay primer mix:

[0080] After diluting the three primers of AX-95000860 to 100 μM with Tris HCl, according to the volume ratio AX-95000860-KASP-FAM_C-R:AX-95000860-KASP-HEX_T-S:AX-95000860-KASP-R:Tris HCl=6 : 6: 15: 23 ratio, aliquoted and stored at -20°C, used as KASP assay primers.

[0081] The dilution and mixing method of AX-94638908 primer is the same as AX-95000860.

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Abstract

The invention discloses two single nucleotide polymorphism molecular markers which can be used for high-throughput marker-assisted breeding of a powdery mildew resistance gene Pm5e and an applicationmethod of the markers in breeding. Pm5e is highly resistant or immune to current prevailing powdery mildew microspecies in China and has an important utilization value in breeding. These two detectionmarkers are KASP markers, can quickly and efficiently achieve high-throughput detection of the distribution of the wheat powdery mildew resistance gene Pm5e in wheat varieties and the distribution ofthe wheat powdery mildew resistance gene Pm5e in isolated populations, facilitate fine locating and cloning of Pm5e and can achieve marker-assisted selection of Pm5e in breeding populations. By meansof Pm5e, the powdery mildew resistance of wheat is improved, the impact on wheat yield is reduced, and the breeding efficiency of the wheat with powdery mildew resistance is improved.

Description

technical field [0001] The invention relates to the technical field of crop molecular marker-assisted breeding, in particular to a high-throughput detection marker of wheat powdery mildew resistance gene Pm5e and its application in breeding. Background technique [0002] Wheat (Triticum aestivum L.) is the second largest food crop after rice in my country, with an annual planting area of ​​more than 26.6667 million mu, accounting for about 27% of the total area of ​​food crops. Therefore, ensuring the steady improvement of wheat yield and quality is an important factor related to the continuous improvement of my country's food security and people's living standards. [0003] Wheat powdery mildew is a worldwide disease, distributed all over the world, especially in areas with high humidity. When the disease occurs, the production is generally reduced by 5%-10%, and the production of seriously diseased fields is reduced by more than 20%. In recent years, the incidence range ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘树兵袁秀芳王洪刚
Owner SHANDONG AGRICULTURAL UNIVERSITY
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