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A marker primer and application of wheat powdery mildew resistance gene stpk-v

A powdery mildew resistance gene and wheat powdery mildew technology, applied in the field of agricultural biology, can solve the problems of no research on marker amplification and achieve good stability

Active Publication Date: 2018-05-01
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, some scholars have screened the available Pm21 Molecular markers of genes, such as OPH17 1900 (Qi LL, Cao MS, Chen PD , Li WL, Liu DJ. Identification, mapping and application of polymorphic DNA associated with resistance gene Pm21 of wheat. Genome ,1996,39: 191-197), OPH17 1400 、SCAR 1265 、SCAR 1400 (Liu Zhiyong et al., Wheat Powdery Mildew Resistance Gene Pm21 Molecular identification and marker-assisted selection of SCAR markers. Acta Genetica Sinica, 1999, 26 (6): 673-682; Liu Z, Sun Q, Yang T. Development of SCAR markers linked to the Pm21 gene conferring resistance to powderymildew in common wheat. Plant breeding 1999, 118, 215-219), but in the screening process of these markers, the researchers only analyzed the polymorphism between materials containing chromosome 6V and common wheat, no Investigating the amplification of these markers in materials involving the other six chromosomes of T. villosa, it was subsequently found that the molecular marker OPH17 1900 and OPH17 1400 The target bands were also amplified in the materials involved in other chromosomes (other than chromosome 6V) of A. villosa, indicating that neither of these two molecular markers were markers for chromosome 6VS specialization of A. t. villosa; the primers designed by Cao Aizhong et al. using the probe Contig17515 (NAU / xibao15F and NAU / xibao15R) were amplified by PCR, and PCR bands of different sizes could be amplified from 6AS, 6BS, 6DS of common wheat and 6VS of T. Pm21 Linked co-dominant molecular marker NAU / xibao15 902 (Cao AZ, Wang XE, Chen YP, Zou XW, Chen PD. A sequene-specific PCR marker linked with Pm21 distinguishes chromosomes 6AS, 6BS, 6DS of Triticum aestivum and 6VS of Haynaldia villosa . Plant Breeding , 2006, 125:201-205), the marker contains Pm21 In the 6VS translocation line of the gene, a specific band of 902bp can be amplified, which is a marker for the chromosome 6VS specialization of T. villosa, but 5 and 4 bands of this marker were amplified in powdery mildew resistant and susceptible plants respectively Bands, difficult to distinguish during electrophoresis
[0005] InDel molecular marker is a detection method for detecting the insertion or deletion in the DNA segment by PCR to find the linkage with the target gene. InDel widely exists in different plant individuals. With the completion of the whole genome sequencing of some important crops and the development of the second generation sequencing technology Widely used, using improved bioinformatics methods, a large number of InDels that can be used to develop molecular markers can be mined in different genotypes. In theory, there is an InDel site near or inside each gene on average, and these InDels can be located in the gene The interregion can also be located in the promoter region, exon region, intron region and 3', 5'UTR region of the gene (Zhang Tifu et al., Mining of maize functional Insertion / Deletiion (InDel) molecular markers and their hybridization application in the identification of species purity. Maize Science, 2012, 20 (2): 64-68), but InDel is applied to Stpk-V Genetic testing has not yet been reported

Method used

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  • A marker primer and application of wheat powdery mildew resistance gene stpk-v
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  • A marker primer and application of wheat powdery mildew resistance gene stpk-v

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Labeled primer InDel w190 with NAU / xibao15 902 Comparative experiment in different wheat genetic materials

[0030] Using the DNA of common wheat China Spring, Ningmai No. 9, common wheat-M. w190 Primers for PCR amplification.

[0031] The PCR reaction system is 25 µL, containing 40 ng of DNA template, 5 pmol of each primer of SEQ ID NO: 1 and SEQ ID NO: 2, 1×PCR buffer, 1.5 mM MgCl 2 , 0.2 mmol dNTP, Taq DNA polymerase 1U;

[0032] The PCR reaction cycle program is: pre-denaturation at 94°C for 3min, denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, 35 cycles; extension at 72°C for 10min; storage at 4°C;

[0033] The PCR product was electrophoresed with 1% Metaphor agarose gel, the electrophoresis voltage was 100v, and the electrophoresis time was 2h. The results were as follows: figure 1 As shown, among them, lane M is the molecular weight marker, lane 1 is common wheat Chinese spring, lane 2 is Ningmai No. 9, lane 3 i...

Embodiment 2

[0035] Example 2 Labeled primer InDel w190 Analysis in the Heterochromatic System of Common Wheat-Tuviflorum 6V

[0036] Using DNA from different wheat materials as templates, InDel w190 As primers, carry out PCR amplification and electrophoresis to the wheat of common wheat-Tambulina villosa 6V heterochromatic system, the PCR reaction system and electrophoresis are the same as in Example 1, and the electrophoresis results are as follows: image 3 As shown, lane M is the molecular weight marker, lane 1 is the common wheat-P. Lane 5 is Zhengmai 883, lane 6 is 06R33, lane 7 is 06R41, lane 8 is del6VS-1, lane 9 is 92R139, lane 10 is 92R90, lane 11 is YC138-4, lane 12 is NJ4, lane 13 is YC24 , lane 14 is NJ1-15, and lane 15 is YC146-4; the results show that the specific band with a size of 190bp and 199bp band, while only a 199bp band could be amplified in common wheat China spring, which is susceptible to powdery mildew.

Embodiment 3

[0038] Labeled Primer InDel w190 F hybridization of Ningmai 9 with the wheat-T. villosa 6VS small fragment translocation line YC72-2 2 Validation in generation plants

[0039] Take the F of Ningmai No. 9 × YC72-2 2 18 plants in the generation population and the DNA of 2 parents are used as templates for PCR amplification, and the PCR reaction system and electrophoresis are the same as in Example 1, and the electrophoresis results are as follows: Figure 4 As shown, where M is the molecular weight marker, lane 1 is the parent YC72-2, lane 2 is the parent Ningmai No. 9, and lanes 3-20 are F 2 Generation plants, F in lanes 5, 6, 10, 11, 13, 14, 15, 16, 17, 18, 20 2Specific bands of 190bp and 199bp were amplified from the generation plants and the parent YC72-2 of lane 1, which were resistant to powdery mildew. F 2 Only 199bp band was amplified in Ningmai 9, the single plant of the generation and the parent of Lane 2, which was a plant susceptible to powdery mildew.

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Abstract

The present invention provides a marker primer of wheat powdery mildew resistance gene Stpk-V and its application in the detection of resistance to wheat powdery mildew Stpk-V gene, the marker primer InDelw190 consists of nucleotide sequence as shown in SEQ ID NO:1 Composition of upstream primers and downstream primers as shown in SEQ ID NO: 2; the present invention is a molecular marker primer developed through the InDel site, which has the advantages of good stability, codominance and easy detection, and can be used on a large scale Molecular marker-assisted selection breeding.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a wheat powdery mildew resistance gene Stpk-V Labeled primers and their applications. Background technique [0002] wheat powdery mildew ( Blumeria graminis f. sp. tritici ) is one of the more and more serious diseases in wheat production. Since the 1980s, powdery mildew has gradually risen from a secondary disease to a major disease in China's main winter wheat areas, and it occurs all year round. From 2004 to 2009, the national average The occurrence area is 6.85 million hm 2 , in the northern winter wheat region and the Huanghuai winter wheat region, powdery mildew is the most important wheat disease, in the middle and lower reaches of the Yangtze River and southwest wheat regions, powdery mildew is the second largest disease after scab or stripe rust. [0003] At present, more than 50 powdery mildew resistance genes have been found in 43 wheat loci, among whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 杨学明吕远大周淼平姚金保杨丹马鸿翔
Owner JIANGSU ACAD OF AGRI SCI
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