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Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof

A resistance gene and molecular marker technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of few molecular markers, the inability to develop functional molecular markers, and the influence of Pi-d2 gene Select efficiency and other issues to achieve the effect of shortening the breeding process and improving breeding efficiency

Active Publication Date: 2013-11-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages: (1) There are relatively few molecular markers that can be developed and utilized; (2) Some genes cannot develop corresponding functional molecular markers
[0009] Although the invention patent with the authorized patent number ZL200310118434.3 discloses a molecular marker dCAPs1 for the Pi-d2 gene, since this marker is only a tightly linked marker of the Pi-d2 gene, it is not a functional molecular marker of the Pi-d2 gene. Therefore, it will undoubtedly affect the selection efficiency of the Pi-d2 gene

Method used

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  • Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof
  • Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof
  • Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Alignment of Pi-d2 allele coding region sequences and identification of SNP

[0041] Design 3 according to the published Pi-d2 gene (GenBank accession number: NM_001064221) (Chen et al., A B-lectin receptor kinase gene conferring rice blast resistance. The Plant Journal, 2006, 46(5): 794-804) For the specific primers (see Table 1), the Pi-d2 gene was amplified from the indica rice variety Gumei 2 with persistent high blast resistance and the rice blast-sensitive japonica variety Nipponbare using PCR amplification technology, and sequenced (the sequencing was entrusted to Shanghai Sonny Company), using the multiple sequence alignment software DNAMAN to compare and analyze the alleles obtained by sequencing, and identified 2 SNPs (single nucleotide polymorphism sites), which are respectively after the translation initiation site Bases 1023 and 2232 were changed.

[0042] Table 1. Amplification-specific primers of Pi-d2 gene

[0043]

Embodiment 2

[0044] Example 2: Design and verification analysis of functional-type specific molecular markers of Pi-d2 gene

[0045] According to the principle of dCAPS marker design (Neff et al., Web-based primer design for single nucleotide polymorphism analysis. Trends Genet.2002,18(12):613-615), using the marker design online software dCAPS Finder2.0 ( http: / / helix.wustl.edu / dcaps / dcaps.html ), corresponding functional molecular markers and their primers were designed for the above two SNPs (see Table 2).

[0046] Table 2. Functional molecular markers of Pi-d2 gene and their primers

[0047]

[0048] Use the two pairs of primers in Table 2 to amplify the disease-resistant (or susceptible) gene in the rice material. After digestion with the restriction endonuclease in Table 2, the material that can be digested contains the resistance gene, and vice versa. for no.

[0049] Utilize above-mentioned synthetic primer to amplify disease-resistant variety and susceptible variety respe...

Embodiment 3

[0057] Example 3: Auxiliary application of functional-type specific molecular markers of Pi-d2 gene in backcross offspring

[0058] Using Digu B, a material with high blast resistance, as the parent, crosses with maintainer lines such as Jin 23B, Zhong 9B, Ganxiang B and Mingzhi B, which are widely used at present, to obtain a batch of traits through multi-generation backcrossing and selfing Excellent maintainer (see Table 3). According to the DNA extraction methods in Wang Ning (2011) (Wang Ning, Molecular marker-assisted selection of Pi5 gene and new material cultivation of blast-resistant rice transfected with sweetpotato storage protein gene, 2011, master's degree thesis, Zhejiang University Library), respectively Extract DNA. The endogenous Pi-d2 gene in the transgenic offspring was analyzed using the functional marker developed in Example 2. The results showed that 50% of the backcross offspring contained the Pi-d2 resistance gene (see Table 3).

[0059] At the same t...

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Abstract

The invention discloses a rice blast resistance gene Pi-d2 functional molecular marker, n exclusive primer sequence thereof, and an application thereof. The functional molecular marker comprises Pi-d2A and Pi-d2B. Pi-d2A is a nucleotide sequence amplified from paddy rice genome DNA with a primer pair SEQ ID NO.1 and SEQ ID NO.2. Through digestion of specific restriction endonuclease MluI, the sequence forms specific band pattern with rice blast resistance gene Pi-d2. Pi-d2B is a nucleotide sequence amplified from paddy rice genome DNA with a primer pair SEQ ID NO.3 and SEQ ID NO.4. Through digestion of specific restriction endonuclease PvuI, the sequence forms specific band pattern with rice blast resistance gene Pi-d2. According to the invention, Pi-d2A and Pi-d2B are amplified from paddy rice by using the primer pairs, and digestion is carried out with corresponding specific restriction endonucleases. At least one of two nucleotide sequences is successfully digested, such that the paddy rice comprises the rice blast resistance gene Pi-d2. According to the invention, resistance gene Pi-d2 in germplasm resources and breeding offsprings can be directly identified, such that seed breeding process is greatly shortened, and seed breeding efficiency is improved.

Description

technical field [0001] The invention relates to the field of rice biotechnology, in particular to a functional molecular marker of rice blast resistance gene Pi-d2 and its special primer sequence and application. Background technique [0002] Rice is one of the main food crops that humans depend on for survival, and it is the staple food for more than half of the world's population. Rice blast is one of the most devastating fungal diseases of rice production in the world (Ou et al. Rice diseases (2nd edition). The Cambridge News Ltd, Cambridge, UK, 1985, 380-381). So far, more than 80 countries have reported the occurrence of rice blast, among which Asia and Africa are the most serious. It is estimated that the rice lost to rice blast is enough to feed 60 million people every year (Subhankar et al., Rice blast fungus sequenced. Current Science. 2005, 89(6):930-931). [0003] In agricultural production, the main ways to control rice blast include chemical agent control and ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 潘刚赵晨晨毛节景程方民
Owner ZHEJIANG UNIV
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