Co-dominant Molecular Marker of Brown Planthopper Resistance Gene bph6 in Rice and Its Application

A brown planthopper resistance, molecular marker technology, applied in biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc. Low problems, to achieve the effect of non-destructive detection, convenient and fast detection procedures, and no need for enzyme digestion

Active Publication Date: 2021-11-02
YUAN LONGPING HIGH TECH AGRI CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional rice insect-resistant breeding is phenotypic selection of plants through the identification of insect-resistant traits, which is time-consuming and labor-intensive and is easily affected and limited by environmental conditions. The identification results are prone to errors and the selection efficiency is low.

Method used

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  • Co-dominant Molecular Marker of Brown Planthopper Resistance Gene bph6 in Rice and Its Application
  • Co-dominant Molecular Marker of Brown Planthopper Resistance Gene bph6 in Rice and Its Application
  • Co-dominant Molecular Marker of Brown Planthopper Resistance Gene bph6 in Rice and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Development of co-dominant molecular markers of BPH6 gene

[0038] The rice BPH6 gene is located on the long arm of rice chromosome 4. By amplifying the BPH6 coding region and its upstream and downstream sequences of several known insect-resistant and insect-susceptible rice varieties such as Luoyang 6 (BPH6 introduction line), Huazhan, and IR64, Incremental sequencing, using DNAMAN software and sequence analysis tools such as NCBI database for sequence comparison analysis, it was found that there was a SNP site at 188bp of the nucleotide sequence shown in SEQ ID NO. is T, and the allele of the corresponding susceptible insect is C (such as figure 1 shown in A). In addition, it is also found that there is a specific coding sequence in the 4438bp-4445bp of the nucleotide sequence shown in SEQ ID NO.1 in the BPH6 gene, wherein the BPH6 insect-resistant allelic nucleotide sequence is 5'-CGAAACAC-3', The allelic nucleotide sequence corresponding to susceptible i...

Embodiment 2

[0039] Example 2 Design of detection primers for co-dominant molecular markers of BPH6 gene

[0040] Detection primers were designed for the two BPH6 gene-specific molecular markers developed in Example 1, and the principles of primer design were as follows.

[0041] First, design specific primers for amplifying the insect-resistant alleles, and use the complementary sequence 5'-GTGTTTCG-3' of the BPH6 insect-resistant allele-specific sequence 5'-CGAAACAC-3' as the 3' end of the primer to design the reverse primer B6 -RR, then design the paired forward primer B6-RF on its upstream, the primers RR and RF can only specifically amplify the BPH6 resistant allele, and produce a 283bp band, while the BPH6 susceptible allele can only amplify Increase time without belt;

[0042] Then, design specific primers for amplifying the susceptible allele, with the C base unique to the susceptible allele in the SNP at position 188 in the coding region of the gene as the 3' end of the primer, a...

Embodiment 3

[0046] Example 3 Establishment of detection method for specific molecular marker of rice brown planthopper resistance gene BPH6

[0047] According to the detection primers of two pairs of molecular markers of BPH6 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:

[0048] 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, 0.1 μL Taq DNA polymerase; 2 μL DNA template, double distilled water to make up the remaining quantity.

[0049] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°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 present invention provides rice brown planthopper resistance gene BPH6 co-dominant molecular marker and application thereof, said molecular marker is a nucleotide sequence containing the 188th polymorphism as shown in SEQ ID NO.1 as T / C, and / or, it is a nucleotide sequence containing the polymorphism 5'-CGAAACAC-3' / 5'-TGCAGGGG-3' as shown in SEQ ID NO.1 at positions 4438-4445. Specific amplification primers were designed for the molecular markers, and PCR amplification was used to detect the BPH6 genotype. The molecular marker of BPH6 and its amplification primers provided by the present invention can be used to identify the genotype of rice BPH6 and to breed rice resources resistant to brown planthoppers. It has the advantages of high identification accuracy, simple operation, low cost, etc., and can shorten the breeding time of rice resistant to brown planthoppers. Cycle, reduce breeding costs.

Description

technical field [0001] The invention relates to the fields of molecular biology and plant molecular breeding, in particular to a co-dominant molecular marker of the rice brown planthopper resistance gene BPH6 and its application. Background technique [0002] Brown planthopper (Nilaparvata lugens Stal, referred to as BPH) belongs to Homoptera planthopper insects, is the main pest of rice. The brown planthopper sucks the rice vascular bundle sheath juice through the needle-like mouthparts, causing the rice plants to turn yellow, lodging or even die. Brown planthoppers spread rice grassy dwarf disease and leaf dwarf disease when they feed, and also promote the spread of rice sheath blight and sclerotinia sclerotiorum. With the popularization of high-yield and non-insect-resistant rice varieties, as well as changes in rice planting methods and farming systems, the occurrence frequency of brown planthoppers has increased and the degree of damage has increased. It has become the...

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 杨远柱邓钊何光存郭建平王凯秦鹏符辰建刘开雨
Owner YUAN LONGPING HIGH TECH AGRI CO LTD
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