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Functional molecular marker of rice nilaparvata lugens resistance gene Bph9, identification method and application

A brown planthopper resistance and functional molecule technology is applied in the fields of rice resistance breeding and molecular genetics, which can solve the problems of limiting the application of Bph9 gene, affecting the efficiency and accuracy of molecular marker-assisted breeding selection, and difficulty in accurately distinguishing rice brown planthopper resistance genes. Achieve the effect of low cost and simple identification operation method

Active Publication Date: 2019-05-17
江西省农业科学院水稻研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is: it is difficult to accurately distinguish rice brown planthopper resistance genes in the prior art by using linkage markers, which affects the selection efficiency and accuracy of molecular marker-assisted breeding, and limits the use of Bph9 gene in the rice brown planthopper resistance breeding process. To provide a functional molecular marker, identification method and application of rice brown planthopper resistance gene Bph9

Method used

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  • Functional molecular marker of rice nilaparvata lugens resistance gene Bph9, identification method and application
  • Functional molecular marker of rice nilaparvata lugens resistance gene Bph9, identification method and application
  • Functional molecular marker of rice nilaparvata lugens resistance gene Bph9, identification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Identification method of molecular marker Bph9-M of rice brown planthopper resistance gene Bph9

[0039] A method for identifying the molecular marker Bph9-M of the rice brown planthopper-resistant gene Bph9, the identification method comprising the following steps:

[0040] RIGW searches for the gene sequence of this site in indica rice, which is Zhenshan 97 (OsZS_12T0336600) and / or Minghui 63 (OsMH_12T0305200); MSU searches for the gene sequence of this site in japonica rice, and the japonica rice is Nipponbare (LOC_Os12g37280) ; The gene sequence Bph9 (ID No. KU216221) and its allelic sequences Bph1 (ID No. KX681949), Bph7 (ID No. KU221258), Bph10 (ID No. KX681950), and Bph21 (ID No. KX681951) were obtained from NCBI Genbank.

[0041] After comparing multiple sequences together, select the Indel sequence that exists in the third exon. The online primer design software Primer1 was used to design molecular markers, named Bph9-M, forward primer: TCATTAGGAACA...

Embodiment 2

[0053] Embodiment two: mark Bph9-M to the detection of multiple different rice varieties

[0054] 1. Test materials

[0055]

[0056]

[0057] 2. DNA extraction

[0058] In this experiment, the CTAB simple method was used to extract genomic DNA from rice samples, and the specific steps were as follows:

[0059] (1) Cut about 0.5g leaves into pieces and put them into a 2ml centrifuge tube, then put a steel ball into the tube, cover the tube cap, add 600μl CTAB extract into the centrifuge tube, fix it on the proofer at 23~ Vibrate at a speed of 26 rev / S for about 60s.

[0060] (2) Water bath at 65°C for 30 minutes, during which time shake and mix twice.

[0061] (3) Open the centrifuge tube, add 600 μl chloroform, shake well, let stand for 5 minutes, and centrifuge at 12000 rpm for 8 minutes.

[0062] (4) Pipet 400 μl of supernatant into a 1.5ml centrifuge tube prepared in advance, add 1 mL of absolute ethanol pre-cooled in a -20°C refrigerator, shake well, let stand i...

Embodiment 3

[0067] Embodiment three: Luojia No. 9 / Ganxiang B and F thereof 2 Detection of Bph9-M in populations

[0068] 1. Test materials

[0069]Luojia 9, Ganxiang B, and F prepared with Luojia 9 / Ganxiang B 2 Segregated populations (c-y) were used for validation of marker Bph9-M.

[0070] 2. DNA extraction

[0071] In this experiment, the CTAB simple method was used to extract genomic DNA from rice samples, and the specific steps were as follows:

[0072] (1) Cut about 0.5g leaves into pieces and put them into a 2ml centrifuge tube, then put a steel ball into the tube, cover the tube cap, add 600μl CTAB extract into the centrifuge tube, fix it on the proofer at 23~ Vibrate at a speed of 26 rev / S for about 60s.

[0073] (2) Water bath at 65°C for 30 minutes, during which time shake and mix twice.

[0074] (3) Open the centrifuge tube, add 600 μl chloroform, shake well, let stand for 5 minutes, and centrifuge at 12000 rpm for 8 minutes.

[0075] (4) Pipet 400 μl of supernatant into...

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Abstract

The invention relates to the technical field of rice resistance breeding and molecular genetics, and particularly discloses a functional molecular marker of a rice nilaparvata lugens resistance gene Bph9, an identification method and application. The molecular marker is Bph9-M, and the nucleotide sequence thereof is shown in a sequence table SIPOSequenceListing 1.0; a Bph9-M primer is a forward primer TCATTAGGAACAGGCTATGCA and a reverse primer TTCTTGTTGACACCGCTCAC. The application of the molecular marker Bph9-M in the breeding of rice nilaparvata lugens resistance includes the following stepsof extracting rice genomic DNA, conducting PCR amplification detection for genotype identification and conducting rice nilaparvata lugens resistance phenotype identification. The molecular marker caneffectively distinguish the Bph9 from other alleles of Bph1, Bph2, Bph7, Bph10, Bph18, Bph21 and the like, be a PCR type intronic functional molecular marker designed for the Bph9 gene, and can carryout rice breeding practice in a simple, rapid and high-throughput manner.

Description

technical field [0001] The invention belongs to the fields of rice resistance breeding and molecular genetics, and specifically relates to a functional molecular marker, identification method and application of rice resistance gene Bph9 to brown planthopper. Background technique [0002] The brown planthopper is one of the most serious pests to rice yield. In addition, brown planthoppers feed on and spread rice diseases, such as tussock dwarf disease and leaf dwarf disease, which indirectly aggravate the damage to rice. At present, the control of brown planthoppers is mainly based on chemical pesticides. The widespread use of insecticides has alleviated the harm of brown planthoppers to a certain extent, but the excessive use of insecticides has caused environmental pollution, pesticide residues, destruction of ecological balance, and drug resistance. issues such as sex. Many practices have shown that using rice's own resistance to breed rice varieties resistant to brown p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11A01H1/04
Inventor 陈明亮肖叶青罗世友沈雨民胡兰香熊焕金
Owner 江西省农业科学院水稻研究所
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