Co-dominant molecular marker of rice blast resistance gene pi2 and its detection method and application

A technology for resistance to rice blast and rice, applied in the field of molecular genetics and breeding, can solve the problems of cumbersome dCAPS or CAPS marker detection methods, restricting the efficient utilization of Pi2 gene, and prone to recombination and exchange of linked markers, so as to achieve non-destructive detection, rapid Detection, low cost effect

Active Publication Date: 2022-05-06
YUAN LONGPING HIGH TECH AGRI CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some PCR-based and Pi2-linked markers or Pi2-based CAPS or dCAPS markers have been developed for the rice blast resistance gene Pi2. These markers provide convenience for Pi2 in molecular breeding, but there are also many shortcomings, such as linked markers. Prone to recombination exchange, dCAPS or CAPS marker detection methods are relatively cumbersome, and the detection cost is relatively high, which limits the efficient use of Pi2 gene in the selection of rice blast resistance varieties

Method used

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  • Co-dominant molecular marker of rice blast resistance gene pi2 and its detection method and application
  • Co-dominant molecular marker of rice blast resistance gene pi2 and its detection method and application
  • Co-dominant molecular marker of rice blast resistance gene pi2 and its detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Development of Co-dominant Molecular Marker of Rice Blast Resistance Pi2 Gene

[0048] The rice blast resistance gene Pi2 is located on chromosome 6 of rice. Through the analysis of Pi2 gene and its multiple alleles Pi9, Piz-t donor rice parent materials C101A51, Toride-1, 75-1-127, Gumei 4 No., kasalath, 638S, 02428, Huazhan, Nipponbare, 9311 and more than 20 rice materials were resequenced, and sequence comparison analysis was performed using software such as DNAMAN, and a specific nucleotide sequence was found in the Pi2 gene sequence ( Located at the 10389729-10389747bp of the sixth chromosome of Nipponbare rice), in which Pi2 is 5'-CAGGAATCTCAGATGG-3', and the other alleles are 5'-TGTTATTCTCAGGTATTAT-3' (the sequence alignment results of some rice materials are as follows figure 1 shown); in addition, a specific SNP site was found downstream of the Pi2 gene (located at the 10452027bp of the 6th chromosome of Nipponbare rice), wherein Pi2 was A, and the ot...

Embodiment 2

[0049] The detection primer design of embodiment 2Pi2 gene co-dominant molecular marker

[0050] Design detection primers for the two Pi2 gene-specific molecular markers developed in Example 1, the primer design principles are as follows:

[0051] First, design and amplify Pi2 disease-resistant allele-specific primers, and use the specific nucleotide sequence 5'-CAGGAATCTCAGATGG-3' in the Pi2 gene as the 3' end of the primer to design the upstream primer Pi2-RF (in order to improve primer specificity and optimize amplification. effect, carry out individual base substitutions), and then design the reverse primer Pi2-RR downstream of it. The primers Pi2-RF and Pi2-RR can only specifically amplify the Pi2 allele and produce a 550bp band, while the other alleles have no band;

[0052] Then design specific primers for amplifying the remaining alleles, design the reverse primer Pi2-SR with the C base complementary to G in the specific SNP site downstream of the Pi2 gene as the 3' e...

Embodiment 3

[0056] Example 3 Establishment of rice blast resistance gene Pi2-specific molecular marker detection method

[0057] According to the detection primers of the two molecular markers of Pi2 designed in Example 2, the reaction program and reaction system of PCR were designed, and through continuous optimization, the following reaction program and system were determined:

[0058] PCR reaction system (10 μL): record as: 10×PCR reaction buffer 1 μL, 10 mM dNTP 0.8 μL, 4 primers (10 μM) 0.15 μL, Taq DNA polymerase 0.1 μL; DNA template 2 μL, double distilled water to make up the remaining quantity.

[0059] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 45 seconds, a total of 35 cycles; extension at 72°C for 8 minutes.

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Abstract

The invention relates to a co-dominant molecular marker of rice blast resistance gene Pi2, a detection method and application thereof. The co-dominant molecular markers of the rice blast resistance gene Pi2 include the location at 10389729-10389747bp of rice chromosome 6, the polymorphism is 5'‑CAGGAATCTCAGATGG‑3' / 5'‑TGTTATTCTCAGGTATTAT‑3' and the location at rice number 6 At chromosome 10452027bp, the polymorphism is the molecular marker of A / G. The molecular marker provided by the present invention can effectively identify Pi2 from other multiple alleles, and has a high degree of specificity and accuracy; the detection method of the Pi2 genotype provided by the present invention determines the rice to be tested by the electrophoresis band of the amplified product The Pi2 genotype of the material does not need enzyme digestion, and the cost is low, which is conducive to shortening the breeding cycle of rice and reducing the breeding cost.

Description

technical field [0001] The invention relates to the field of molecular genetic breeding, in particular to a co-dominant molecular marker of rice blast resistance gene Pi2, a detection method and application thereof. Background technique [0002] Rice is one of the most important food crops in the world, and nearly half of the population uses rice as a staple food. Rice blast is one of the most serious diseases in rice production, which poses a major threat to rice safety production. Long-term production practices have shown that cultivating rice resistant varieties or germplasm is one of the effective measures to control rice blast. However, due to the frequent pathogenic variation of rice blast physiological races, the resistance of single-resistant varieties will gradually lose after planting for 3 to 5 years. Therefore, mining and utilizing spectral resistance genes is the key to obtaining durable resistant varieties important way. In recent decades, many rice blast re...

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 杨远柱邓钊王凯赵炳然刘兰兰石媛媛秦鹏符辰建
Owner YUAN LONGPING HIGH TECH AGRI CO LTD
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