Functional specific molecular marker PikFNP for resistance genes Pik of rice blast and method and application of functional specific molecular marker PikFNP

A resistance gene and molecular marker technology, applied in the field of agricultural biology, can solve the problems of error in identification results and low selection efficiency of target genes, and achieve the effect of reducing labor costs and improving the efficiency of breeding work.

Inactive Publication Date: 2012-08-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional rice resistance breeding relies on the identification of resistance phenotypes, which not only requires breeders to have rich experience in inoculation and investigation, but is also easily affected by environmental and human factors, and the identification results are prone to errors. The selection of target genes tends to be less efficient

Method used

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  • Functional specific molecular marker PikFNP for resistance genes Pik of rice blast and method and application of functional specific molecular marker PikFNP
  • Functional specific molecular marker PikFNP for resistance genes Pik of rice blast and method and application of functional specific molecular marker PikFNP
  • Functional specific molecular marker PikFNP for resistance genes Pik of rice blast and method and application of functional specific molecular marker PikFNP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Alignment of Pik allele coding region sequence and SNP identification

[0040] Amplification by conventional PCR (Yuan et al. 2011. The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NB S-LRR genes. Theor Appl Genet 122: 1017-1028; Zhai et al. al.2011.The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication.New Phytologist189:321-334), sequencing (Shanghai Yingjun Biotechnology Co., Ltd., Guangzhou Branch), from 5 disease resistance Rice variety Shin 2 (Pik-s donor variety; Kiyosawa.1987. With genetic view on the mechanism of resistance and virulence. Japanese Journal of Genetics 41: 89-92), Tsuyuake (Pik-m donor variety; Ashikawa et al .2008.Two adjacent nucleotide-binding site-leucine rich repeat class genes are required to confer Pikm-specific rice blast resistance.Genetics180: 2267-2276), Kusabue (Pik donor variety; Zhai et al.2011. The isolation and characteriza...

Embodiment 2

[0042] Example 2: Pik functional specific molecular marker and its primer design and detection

[0043] According to the principle of dCAPS labeling (Neff et al., 2002. Web-based primer design for the single nucleotide polymorphism analysis. Trends in Genetics 18: 613-615), the functional specificity is designed at 100-200bp upstream of the above SNP site The upstream primer Pik-FNP-F is shown in SEQ ID NO.1; the functionally specific downstream primer Pik-FNP-R with mismatched bases is designed at the above SNP site, as shown in SEQ ID NO.2 . Use this set of primers to amplify the DNA template of the variety carrying the Pik gene and other alleles.

[0044] The PCR amplification reaction system of Pik-FNP is as follows:

[0045]

[0046] Pik-FNP PCR amplification reaction temperature cycle conditions are as follows: 94°C for 3 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C for 5 minutes; 10°C storage.

[0047] After the PCR reaction is complet...

Embodiment 3

[0050] Example 3: Application of specific molecular markers of resistance gene Pik in identifying different rice blast resistance genes

[0051] Extracted 12 rice varieties containing different rice blast resistance genes [respectively: Kusabue, Kando51, Kuyuku 131, GA20, Taifenzhan (Zhai et al.2011. The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication.New Phytologist 189:321-334), Tsuyuake(Ashikawa et al.2008.Two adjacent nucleotide-binding site-leucine rich repeat class genes are required to confer Pikm-specific rice blast resistance.Genetics 180:2267-2276) , K60(Yuan et al.2011.The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes.Theor Appl Genet 122: 1017-1028), IRBLk-Ka, IRBLKm- Genomic DNA of TS, IRBLkp-K60, IRBLks-S, and IRBLKh-K3 (Kobayashi et al. 2007. Development of new sets of international standard differential varieties for blast resistance in rice (Oryza ...

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Abstract

The invention discloses a functional specific molecular marker PikFNP for resistance genes Pik of rice blast and a method and application of the functional specific molecular marker PikFNP. The molecular marker Pik-FNP is a nucleotide sequence which is amplified from total deoxyribonucleic acid (DNA) of rice by a primer pair SEQ ID NO. 1 and SEQ ID NO. 2. By a method for contrasting an allelic gene sequence of multiple resistance genes Pik of rice blast to a Pik-1 sequence and a Pik-2 sequence, Pik functional specific single nucleotide polymorphism (SNP) which can be different from susceptible allelic genes and other resistance genes of rice blast can be obtained through analysis, and a primer SEQ ID NO. 1 and a primer SEQ ID NO. 2 are obtained by design; and the DNA of rice is subjected to amplification and specific digestion so as to obtain the Pik functional specific molecular marker Pik-FNP. The Pik functional specific molecular marker Pik-FNP can be screened from a large number of rice genetic resources and can be applied to molecular marker-assisted selection breeding, gene aggregation breeding and transgenic breeding, and the functional resistance genes can be identified.

Description

Technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a functional molecular marker PikFNP of the rice blast resistance gene Pik, and a method and application thereof. Background technique [0002] Rice is one of the most important food crops in the world. About half of the population feed on rice as the staple food. Rice blast, caused by Magnapothe oryzae, is one of the most serious diseases to rice production, causing serious food losses every year. From the point of view of environmental protection and sustainable agricultural development, the breeding and utilization of disease-resistant varieties is the safest and most effective way to control rice blast. Traditional rice resistance breeding relies on the identification of resistance phenotypes, which not only requires breeders to have rich experience in inoculation and investigation, but is also vulnerable to environmental and human factors. The identification...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 潘庆华翟纯林菲王玲
Owner SOUTH CHINA AGRI UNIV
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