Functional specificity molecule marker Pi7FNP of blast-resistance gene Pi7, method and application thereof

A resistance gene and molecular marker technology, applied in the field of agricultural biology, to improve breeding efficiency and reduce labor costs

Inactive Publication Date: 2012-10-17
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the fine mapping of Pi7, the applicant found that in the genomic region containing the Pi7 site, the Pi7 donor variety IRBL7-M has a similar genome structure to the Pik-m, Pik and Pik-p donor varieties, that is, the There are large insertions / deletions in the reference sequence Nipponbare

Method used

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  • Functional specificity molecule marker Pi7FNP of blast-resistance gene Pi7, method and application thereof
  • Functional specificity molecule marker Pi7FNP of blast-resistance gene Pi7, method and application thereof
  • Functional specificity molecule marker Pi7FNP of blast-resistance gene Pi7, method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Alignment of Pi7 allele coding region sequences and identification of SNP

[0071] Through conventional PCR amplification (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), sequencing (Shanghai Yingjun Biotechnology Co., Ltd., Guangzhou Branch), from four rice varieties IRBL7-M (Development of co-dominant amplified polymorphic sequence markers in rice that flank the Magnaporthe grisea gene Pi7(t) in recombinant inbred line 29, Compbell et al.2004.Phytopathology 94:302-307), K60 (Pik-p donor species; same as above, Yuan et al.2011.Theor Appl Genet 122:1017-1028), Kusabue (Pik donor species; Zhai et al. al.2011.The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication.New Phytologist 189:321-334) and Tsuyuake (Pik-m donor species; Ashikawa et al.2008.Two adjacent nucleotide- Binding site-leucine-rich r...

Embodiment 2

[0072] Example 2: Pi7 function-specific molecular markers and their primer design and detection

[0073]According to the design principle of dCAPS markers (Derived Cleaved Amplified Polymorphic Sequences; Neff et al.2002.Web-based primer design for single nucleotide polymorphism analysis.Trends in Genetics 18:613-615), Primer Premier 5.0 software tool (http: / / www.premierbiosoft.com), designed a functional specific upstream primer Pi7-1FNP-F at 100-200bp upstream of Pi7-1SNP, and designed a functional specific downstream primer Pi7-1FNP-R with mismatched bases at the SNP , respectively as shown in SEQ ID NO.1 and SEQ ID NO.2; respectively design functional specific upstream primer Pi7-2FNP-F and functional specific downstream primer Pi7-2FNP within the range of 100bp upstream and downstream of Pi7-2 SNP site -R, respectively shown in SEQ ID NO.3 and SEQ ID NO.4. The DNA templates of varieties carrying the Pi7 gene and other alleles were amplified by these two sets of primers,...

Embodiment 3

[0104] Example 3: Application of the resistance gene Pi7 function-specific molecular marker in the identification of different rice blast resistance genes

[0105] Seven rice varieties containing different rice blast resistance genes were collected, namely: IRBL7-M, IRBLk-Ka, IRBLkp-K60, IRBLKM-TS (Kobayashi et al.2007.Development of new sets of international standard differential varieties for blast resistance in rice (Oryza sativa L.).JARQ ​​41:31-37), and the genomic DNA of Q1063 and Longjing 24 (Longjing24) (supra, Zhai et al.2011.New Phytologist 189:321-334), and Using this as a template, using the PCR amplification reaction system and reaction conditions of Pi7-1FNP and Pi7-2FNP described in Example 2, PCR amplification, enzyme digestion and electrophoresis detection were performed on the molecular marker specific to the function of the resistance gene Pi7. The electrophoresis results were as image 3 A and image 3 Shown in B.

[0106] As illustrated in the accompany...

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Abstract

The invention discloses a functional specificity molecule marker Pi7FNP of blast-resistance gene Pi7, a method and an application thereof. The molecule marker consists of Pi7-1FNP and Pi7-2FNP, wherein Pi7-1FNP is the nucleotide sequence amplified from rice total DNA by SEQ ID NO.1 and SEQ ID NO.2, and Pi7-2FNP is the nucleotide sequence amplified from rice total DNA by SEQ ID NO.3 and SEQ ID NO.4. According to the invention, two single-base differences which can be different from the Pi7 functional specificity of the disease-susceptibility alleles and other blast-resistance genes are gained by the analysis through comparing allele sequences of the rice blast-resistance gene Pi7 with the sequences of SEQ ID NO.5 and SEQ ID NO.6; primers SEQ ID NO.1 and primers SEQ ID NO.2 and primers SEQ ID NO.3 and primers SEQ ID NO.4 are respectively obtained by designing; by carrying out DNA amplification and then specific enzyme digestion on rice, the Pi7-1FNP and Pi7-2FNP are respectively obtained. The invention can be applied in screening from large amounts of rice genetic resources and identifying the functional resistant genes, and molecular marker assisted selection, gene polymerization breeding and transgenic breeding.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a rice blast resistance gene Pi7 function-specific molecular marker Pi7FNP and its method and application. Background technique [0002] Rice is one of the most important food crops in the world, and more than half of the population uses rice as a staple food. Rice blast caused by Magnapothe oryzae is one of the most serious diseases to rice production, causing serious grain loss every year. From the point of view of environmental protection and agricultural sustainable 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 easily affected by environmental and human factors. The identificat...

Claims

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Application Information

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