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Function-specific molecular marker of rice blast resistance gene pi64 and its method and application

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: 2015-09-30
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 marker of rice blast resistance gene pi64 and its method and application
  • Function-specific molecular marker of rice blast resistance gene pi64 and its method and application
  • Function-specific molecular marker of rice blast resistance gene pi64 and its method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Pi64 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) Pi64 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 Pi64 The four amino acid sites N698, Y1003, A1031 and V1177 unique to the gene ( figure 1 ).

Embodiment 2

[0048] Example 2: Pi64 Upstream sequence analysis of gene coding region

[0049] Amplified by conventional PCR Pi64 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: Pi64 Development and detection of gene-specific molecular markers

[0051] use exists in Pi64 The specific amino acid site N698 of the gene ( Pi64 The corresponding codon in the 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 as SEQ 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, tak...

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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 present invention belongs to the field of agricultural biotechnology, which involves the resistance gene of rice and rice plague Pi64 (Original number Piym2 ) 2 pairs of functional molecular marks and methods and applications. Background technique [0002] Rice is one of the most important food crops in the world. The population of more than half of the world is the main grain.Rice plague is one of the most important diseases of rice. The global daily production is reduced by 10%to 30%due to rice plague, and it is 30%to 50%in severe times.In my country's rice plague, it has an area of ​​about 3.8 million acres throughout the year. [0003] Cultivating and using disease -resistant varieties have been recognized as the most economical and effective and environmentally friendly rice plastic disease prevention measures.However, due to the complexity of the flock of the rice plague, and its pathogenicity is prone to mutation, some potential or newly generate...

Claims

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Application Information

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Patent Type & Authority Patents(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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