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Function specific molecular markers of rice blast resistance gene Pi63 as well as method and application of function specific molecular markers

A resistance gene and molecular marker technology, applied in the field of agricultural biology, can solve problems such as rice breeding practices that have not yet been applied, and achieve the effects of improving breeding efficiency, wide applicability, and clear breeding selection goals

Active Publication Date: 2014-03-19
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It belongs to a relatively ancient gene in evolution and has not been used in rice breeding practice

Method used

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  • Function specific molecular markers of rice blast resistance gene Pi63 as well as method and application of function specific molecular markers
  • Function specific molecular markers of rice blast resistance gene Pi63 as well as method and application of function specific molecular markers
  • Function specific molecular markers of rice blast resistance gene Pi63 as well as method and application of function specific molecular markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Pi63 Amino acid sequence alignment of its alleles and identification of specific amino acid sites

[0047] Through PCR amplification and sequencing methods, from Wool Valley (YMG), Qitou Baigu, 93-11 (Yangdao No. Genomic DNA obtained from Nong 363 and Lijiang Xintuan Heigu (LTH) Pi63 DNA sequence of allele coding region, 3 genes of reference variety Nipponbare obtained from rice database LOC_Os01g57280 , LOC_Os01g57310 and Pish DNA sequence of the coding region. Using the software tool Primer Premier 5 to translate the DNA sequence into an amino acid sequence, and then using the software tool to perform multiple sequence alignment analysis on the obtained amino acid sequence, it was identified that Pi63 The four amino acid sites N698, Y1003, A1031 and V1177 unique to the gene ( figure 1 ).

Embodiment 2

[0048] Example 2: Pi63 Upstream sequence analysis of gene coding region

[0049] Amplified by conventional PCR Pi63 The upstream sequence of the coding region of the gene was sequenced to obtain a 1597bp sequence. Through NCBI BLAST comparison (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&BLAST_ PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome), a 715bp TIR type was found upstream of the coding region transposon, but no homologous sequence of this transposon was found in rice.

Embodiment 3

[0050] Example 3: Pi63 Development and detection of gene-specific molecular markers

[0051] use exists in Pi63 The specific amino acid site N698 of the gene (the corresponding codon in the Pi63 protein is AAT, and the other varieties are GAT), according to the principle of CAPS marker design, using the online software dCAPS Finder 2.0 (http: / / helix.wustl.edu / dcaps / dcaps .html) and Primer-BLAST (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) to design primer YRT3, the nucleotide sequence of the primer pair is shown in SEQ Shown in ID NO.1 and SEQ ID NO.2. The PCR amplification reaction system of YRT3 is: 2 × PCR Buffer for KOD FX 10 μl, dNTPs (2mM each) 4 μl, primer (10pmol, F+R) 1 μl, KOD FX enzyme (1U / μl, TOYOBO) 0.4 μl , DNA template (100ng / μl) 2 μl, add ddH 2 0 to 20 μl. Amplification reaction conditions: 94°C for 2 min; 98°C for 10 s → 59°C for 1.5 min → 68°C for 5 min, 35 cycles; 68°C for 5 min, stored at 10°C. After the PCR reaction...

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Abstract

The invention discloses two pairs of function specific molecular markers YRT3 and YRT4 of a rice blast resistance gene Pi63 as well as a method and application of the function specific molecular markers. The Pi63 function specific molecular markers YRT3 and YRT4 are obtained by comparing and analyzing Pi63 allelic genes in a plurality of rice varieties and upstream sequences in coding regions thereof to obtain two Pi63 function specific loci different from an infected allelic gene and other rice blast resistance genes; designing a primer to obtain SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; and amplifying total DNA of rice through the primer to obtain Pi63 function specific molecular markers YRT3 and YRT4. The function resistance gene can be screened and identified in a great amount of rice genetic resources, and can be applied to molecular marker assisted selection, genetic pyramiding breeding and transgenic breeding.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, in particular to a rice blast resistance gene Pi63 (Original No. Piym2 ) of 2 pairs of functional molecular markers and their methods and applications. Background technique [0002] Rice is one of the most important food crops in the world, and more than half of the world's population uses rice as the main food. Rice blast is one of the most important diseases of rice. The annual rice production reduction caused by rice blast is 10% to 30%, and in severe cases, it reaches 30% to 50%, or even no harvest. In my country, rice blast occurs in an area of ​​about 3.8 million mu all year round, causing hundreds of millions of kilograms of rice losses and directly threatening the country's food security. [0003] Breeding and utilizing disease-resistant varieties has been recognized as the most cost-effective and environment-friendly control measures for rice blast. However, due to the comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 万建民雷财林马建马小定程治军王久林张欣郭秀平王洁赵志超吴赴清林启冰
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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