High-throughput detection marker of wheat powdery mildew resistance gene pm5e and its application in breeding

A technology for resistance to powdery mildew genes and wheat powdery mildew, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of large genetic distance, long genetic distance, and low detection efficiency.

Active Publication Date: 2021-08-13
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 extent, there was still a long genetic distance , low polymorphism level, low detection efficiency and other problems; Later, Liu Zhiyong et al. (2008) screened specific SNP sites through BSR-Seq, and converted them into specific primers, further Pm5e gene location, the Pm5e The gene was mapped between EST markers CJ729392 and CJ584170, the study improved primer specificity, but the polymorphism level was still low, and the genetic distance between the two markers was large (23.5cM)

Method used

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  • High-throughput detection marker of wheat powdery mildew resistance gene pm5e and its application in breeding
  • High-throughput detection marker of wheat powdery mildew resistance gene pm5e and its application in breeding
  • High-throughput detection marker of wheat powdery mildew resistance gene pm5e and its application in breeding

Examples

Experimental program
Comparison scheme
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) Place young wheat leaves in a 96-well deep-well plate, freeze them with liquid nitrogen and grind them into powder on a tissue grinder; (b) preheat in each well of a 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 gently shake it 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, 4°C for 10min, then take 500μL...

Embodiment 2

[0076] Example 2: Rejuvenation 30 Pm5e Development of tightly linked markers in genes

[0077] At present, 6 have been published with Pm5e Tightly linked markers, 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. Anti-disease material Rejuvenation 30 contains Pm5e gene, Chancellor showed high susceptibility 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-susceptibility gen...

Embodiment 3

[0078] Example 3: Primer dilution and KASP assay primer mix:

[0079] 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.

[0080] 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 of the powdery mildew resistance gene Pm5e that can be used for high-throughput marker-assisted selection breeding and an application method in breeding. Pm5e is highly resistant or immune to the current popular powdery mildew races in my country, and has important utilization value in breeding. These two detection markers are KASP markers, which can quickly, efficiently and high-throughput detect the distribution of the wheat powdery mildew resistance gene Pm5e in wheat varieties and in segregated populations, which is conducive to the fine positioning and cloning of Pm5e, and can Marker-assisted selection was performed on Pm5e in the breeding population, and Pm5e was used to improve wheat powdery mildew resistance, reduce the impact on wheat yield, and improve the efficiency of wheat powdery mildew resistance breeding.

Description

technical field [0001] The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a wheat powdery mildew resistance gene Pm5e High-throughput detection markers and their 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 ​​over 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 o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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