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Specific SNP (Single Nucleotide Polymorphism) co-dominant molecular marker primer in rice brown planthopper resistance gene BPH9 gene and application

A brown planthopper resistance and molecular marker technology, applied in the field of molecular genetics, can solve the problems affecting the efficiency and accuracy of BPH9 selection, and reduce the extension efficiency, and achieve the effects of controlling the size of the breeding population, saving costs, and improving the selection efficiency.

Active Publication Date: 2017-12-19
湖南隆平高科种业科学研究院有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in molecular marker-assisted selection breeding, the selection of BPH9 gene mainly uses markers linked to it, such as RM28483, InD28450, InD28453, InD284323, InD2, InD14, RM28466, RM28481, RM2848, etc., and gene markers have not been reported yet. To a certain extent, it affects the efficiency and accuracy of BPH9 selection in rice breeding for resistance to BPH
[0005] Two-pair cross-primer PCR (PCR with confronting two-pair primers, PCR-CTPP) is a simple, fast and low-cost SNP typing method, which can distinguish 3 Different genotypes, the basic principle is: when there is a mismatch at the 3' of the primer, the extension efficiency will be reduced and the amplification will be hindered

Method used

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  • Specific SNP (Single Nucleotide Polymorphism) co-dominant molecular marker primer in rice brown planthopper resistance gene BPH9 gene and application
  • Specific SNP (Single Nucleotide Polymorphism) co-dominant molecular marker primer in rice brown planthopper resistance gene BPH9 gene and application
  • Specific SNP (Single Nucleotide Polymorphism) co-dominant molecular marker primer in rice brown planthopper resistance gene BPH9 gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Utilize the primers designed by the present invention to amplify 3 kinds of BPH9 genotype DNA templates

[0040] 1) Biomaterials

[0041] The CA-type material is the parental material Pokkali containing the BPH9 gene; the TG-type material is the parental material 9311 containing the susceptible allele; the heterozygous material is the F1 generation material obtained by crossing Pokkali and 9311.

[0042] 2) Rice DNA extraction and primer synthesis

[0043] The DNA of the above materials was extracted by the CTAB method, and the primer sequences shown in Table 1 were synthesized, specifically:

[0044] BPH9-INTF:ACCATTGTTAGGCAGTTGTTCA

[0045] BPH9-EXTR: ATTCGACTCCCTTTTCTTGTTATCT

[0046] BPH9-INTR: CAGCCTCCTGAAGAGATCTTTCA

[0047] BPH9-EXTF:AATGTCGCACCCAGCAGC

[0048] 3)PCR

[0049] The PCR system is recorded as 10ul: 1ul 10×PCR reaction buffer, 0.8ul 10mM dNTP, 0.15ul 10uM primers for all 4 primers, 0.1ul Taq DNA polymerase; 2ul DNA template, double distilled wat...

Embodiment 2

[0053] The invention is used to detect indica and japonica backbone parent materials commonly used in production.

[0054] 1) Biomaterials

[0055] The parental control of CA type is BPH9 donor material Pokkali and 9311 background BPH9 gene introduction line Luoyang 9; the parental control of TG type is 9311. Backbone parent materials: Huazhan, R1212, R1206, Fengyuzhan, 6116-765, R1128, Yuzhenxiang, Huarun 2, CO2, R900, Mf63, Mianhui 3728, Huahui 272, Huahui 19, Huahui 284 , 638S, Nipponbare, Nongken 31, Hujing 6, Hujing 5, Yuejing 0618, 02428, Rejing 35, Zhejing 75, Huijing 602, Zhendao 819, Jiangsu Jing 2, Long 5, Yandao 531, Fukuniski , Yishang S01, Yangjing 5507, Liaoyan 287, Longdao No. 9 (waxy) etc. 34 DNAs were extracted by CTAB method.

[0056] 2) PCR

[0057] See Example 1

[0058] 3) Result analysis

[0059] Such as image 3 As shown, lanes 1 and 2 are the BPH9 donor material Pokkali and the BPH9 introduction line Luoyang 9 both amplified the BPH9 allele-specif...

Embodiment 3

[0061] Detection of single gene isolation of rice brown planthopper resistance gene BPH9F2 population by using the present invention

[0062] 1) Biomaterials

[0063] The F2 segregation population was constructed by crossing the CA-type BPH9 donor material Pokkali with the TG-type susceptible material 9311.

[0064] 2) DNA extraction and PCR

[0065] See Example 1

[0066] 3) Result analysis

[0067] Genotype detection was carried out on individual plants of 96 F2 segregation populations. The segregation ratio of the three different genotypes was 27RR:46H:23S, which was consistent with the Mendelian segregation ratio of 1:2:1 (χ 2 =0.502 0.05 =5.99), therefore, the detected locus showed single-gene segregation (see Figure 4 ).

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Abstract

The invention discloses a specific SNP (Single Nucleotide Polymorphism) co-dominant molecular marker primer in a rice brown planthopper resistance gene BPH9 and belongs to the field of plant molecular breeding. The marker primer and a sequence thereof are shown as follows: BPH9-IF: ACCATTGTTAGGCAGTTGTTCA; BPH9-ER: ATTCGACTCCCTTTCTTGTTATCT; BPH9-IR: CAGCCTCCTGAAGAGATCTTTCA; BPH9-EF: AATGTCGCACCCAGCAGC. The primer disclosed by the invention is used for carrying out PCR (Polymerase Chain Reaction) amplification on a rice DNA (Deoxyribonucleic Acid) sample and can be used for rapidly judging a gene type of rice BPH9 to be detected; the method is simple, convenient and rapid, is low in cost and can be widely applied to molecular marker assisted selective breeding or identification of BPH9 gene types of rice germplasm resources.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and specifically relates to specific SNP co-dominant molecular marker primers in the rice planthopper-resistant gene BPH9 gene and its application. The molecular marker primers provided by the invention are suitable for large-scale screening of BPH9 genotypes in rice breeding And screening new BPH9 genotype resistant parents from rice germplasm resources. Background technique [0002] With the development of biotechnology, human beings have entered the post-genome era, and the comprehensive study of functional genome has become the frontier field of life science research. At present, the fine genetic map and physical map of the rice genome have been completed. Further research on rice functional genes is of great significance to socioeconomic development and biological research. [0003] Rice is an important food crop in my country, and the brown planthopper is one of the most serious pests in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 杨远柱邓钊何光存陈荣智王凯秦鹏符辰建刘开雨
Owner 湖南隆平高科种业科学研究院有限公司
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