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Functional molecular marker for rice anti-blast gene Pi9 and application thereof

A technology for resistance to rice blast and rice, applied in the field of molecular genetics and breeding of crops, can solve the problems of difficulty in accurately identifying disease resistance function genes, prediction error rate, etc., and achieve the effects of high accuracy, good repeatability, and improved breeding efficiency.

Active Publication Date: 2014-12-10
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the results of gene cloning show that disease-resistant genes often exist in clusters, and the sequences between different multiple alleles, between functional sequences and non-functional sequences are highly homologous, and general linkage markers are still difficult to accurately identify each gene. A disease-resistant functional gene, and there is a certain prediction error rate

Method used

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  • Functional molecular marker for rice anti-blast gene Pi9 and application thereof
  • Functional molecular marker for rice anti-blast gene Pi9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Pi9 Functional Gene Sequence Marker Structure Determination

[0031] By analyzing the rice variety 75-1-127 Pi9 Functional gene segment sequence (Genbank DQ454157) and non-functional gene sequence derived from rice variety Nipponbare Pi9 The allelic segment sequence (Genbank DQ454158) found that there were many differences between the two in the promoter region. further targeting Pi9 Design specific primer combination Pro9-F: 5'-CGGTTATAGATGAATATAGTTC-3', Pro9-R: 5'-GTTCGACTCTCCCTTCAAGC-3' for the functional gene promoter -7 to -793 segment, to carry Pi9Genomic DNA of functional gene rice variety 75-1-127 and other 27 rice varieties (Nihonbare, 9311, 02428, BL123, Lijiang Tuanheigu, Katy, Zhen 97A, Longtepu A, Zaoka A, II-32A, Gu Feng A, Tianfeng A, D62A, Gang 46A, Jin 23A, Guangzhan 63S, Minghui 63, Shuhui 527, Minghui 86, ZR101, Guanghui 998, Radiation 838, Ce 64, Miryang 46, SE21S, Pei Dwarf 64S, CO39) genomic DNA was used as a template for PCR amplifi...

Embodiment 2

[0032] Example 2 Pi9 Functional molecular marker establishment

[0033] according to Pi9 Functional gene promoter region and allelic non-functional Pi9 According to the sequencing analysis results of the corresponding segments, a pair of specific primer combinations F9-F: 5′-TGATTATGTTTTTTATGTGGGG-3′, F9-R: 5′-ATTAGTGAGATCCATTGTTCC-3′ were designed. F9-F / F9-R primer combination specific correspondence Pi9 Functional gene promoter -451 to -578 sequence, the amplified fragment includes Pi9 The specific deletion site of functional gene promoter -516 / -517 (attached figure 1 ). 28 rice varieties 75-1-127, Nipponbare, 9311, 02428, BL123, Lijiang Tuanheigu, Katy, Zhen 97A, Longtepu A, Zaoka A, II- 32A, Gufeng A, Tianfeng A, D62A, Gang 46A, Jin 23A, Guangzhan 63S, Minghui 63, Shuhui 527, Minghui 86, ZR101, Guanghui 998, Radiation 838, Measurement 64, Miryang 46, The genomic DNA of SE21S, Pei'ai 64S, and CO39 were amplified by PCR.

[0034] The PCR reaction system was 25 μL, c...

Embodiment 3

[0035] Example 3 Pi9 Verification and application of functional molecular markers

[0036] without Pi9 Nippon Haruwa Pi9 The functional gene donor variety 75-1-127 is used as the parent to configure F 2 population, with Magnaporthe oryzae strain KJ201 on F 2 A group of 247 plants were artificially inoculated at the seedling stage, and 176 resistant plants were obtained. The genomic DNA of these resistant plants was extracted, PCR amplification was performed with the primer combination F9-F / F9-R, and the amplified products were electrophoretically typed on 8% non-denaturing polyacrylamide gel. Example results show that the PCR amplification samples of 176 resistant plants can detect representative Pi9 The 128bp specific band of the functional gene has a selection efficiency of 100%.

[0037] Using the configuration combination of 75-1-127 and rice restorer line Minhui 3301, the Pi9 Functional genes were transferred to Minhui 3301. Matched and screened to obtain BC 3 f ...

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Abstract

The invention provides a functional molecular marker for rice anti-blast gene Pi9 and application thereof, belonging to the field of crop molecular genetic breeding science. The invention finds out that the rice anti-blast gene Pi9 has a 10bp insertion / deletion site positioned between the gene promoter -516 and -517, wherein the nucleotide sequence is disclosed as SEQ ID NO.1; and on such basis, the invention develops a method of the functional molecular marker for gene Pi9. By detecting the functional molecular marker, the invention can accurately detect whether genomes of different rice species contain the Pi9 functional gene and detect the homozygotic state, and can be used for screening rice hybrid transformed descendant plants to enhance the breeding efficiency of the rice anti-blast material, thereby effectively controlling the size of the breeding population, obviously saving the breeding and screening cost and obtaining the anti-blast rice species containing the Pi9 functional gene.

Description

technical field [0001] The invention belongs to the field of crop molecular genetics and breeding, in particular to a rice blast resistance gene Pi9 functional molecular markers and their applications. Background technique [0002] Rice is one of the most important food crops in our country, and it is of vital significance to ensure our country's food security. For a long time, rice production has been seriously threatened by some pests and diseases. Rice blast is one of the most harmful diseases to rice production in the world. Its harm not only reduces rice yield, but also affects rice quality, causing serious losses to rice production. Long-term production practices have shown that breeding and rational use of disease-resistant varieties is the most cost-effective and environmentally safe way to control rice blast. [0003] Over the past 60 years, scientists from various countries have identified more than 70 disease-resistant genes from a variety of rice disease-resis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68A01H1/04
Inventor 陈松彪王锋田大刚陈在杰林艳宋亚娜胡昌泉
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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