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A method and application for creating interspecific hybrid compatibility lines of Asian cultivated rice and African cultivated rice

A technology for cultivating rice and hybrids, which is applied in the field of plant genetic engineering technology and crop genetics and breeding, can solve the problems of no verification, no provision, etc., and achieve the effect of improving rice yield and resistance

Active Publication Date: 2020-12-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the patent publication does not verify ORF12 exist S1 The function of hybrid sterility and whether it is necessary for rice pollen and female gamete development, nor how to create hybrid compatibility lines to overcome interspecific hybrid breeding S1 Hybrid Sterility Techniques

Method used

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  • A method and application for creating interspecific hybrid compatibility lines of Asian cultivated rice and African cultivated rice
  • A method and application for creating interspecific hybrid compatibility lines of Asian cultivated rice and African cultivated rice
  • A method and application for creating interspecific hybrid compatibility lines of Asian cultivated rice and African cultivated rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 OgTPR1 Construction of functional knockout vector

[0031] For Asian-African rice hybrid sterility gene OgTPR1 (SEQ ID NO: 1) coding sequence, design target sequence (SEQ ID NO: 4). Two complementary primers (U3- OgTPR1- F, OgTPR1- R) Formation of the target double-stranded linker (Table 1), ligated into the expression cassette of the sgRNA driven by the rice OsU3 promoter. The PCR-amplified sgRNA expression cassette fragment was cloned into the gene editing binary vector pYLCRISPR / Cas9Pubi-H (contained in the T-DNA region) to generate KO- OgTPR1 ( figure 1 B).

[0032]

Embodiment 2

[0033] Example 2 OgTPR1 Knockout line ogtpr1 Identification

[0034] The binary transformation vector KO- OgTPR1 Agrobacterium-mediated transformation of NIL-g to obtain T 0 transformant. The positive knockout mutant of the transgene was identified by PCR with primers HptF / HptR (Table 1) ( figure 2 A). Further, a fragment containing the target site from two independently transformed plants was amplified by PCR, and sequenced and analyzed OgTPR1 Mutation effect of gene, get mutant ogtpr1-1 with ogtpr1-2 ( figure 2 B). They produce frameshifts and premature stop codons due to single base insertions, i.e. loss-of-function truncated genes ogtpr1, As shown in SEQ ID NO: 2-3.

Embodiment 3

[0035] Example 3 ogtpr1 Knockout mutant T 1 Identification and phenotype observation of plants without transgenic individuals

[0036] to the above ogtpr1 Transgenic T 1 Plants were transgenic ( HPT Gene) PCR detection, isolated individuals without transgene ( image 3 A). Then observe 2 ogtpr1 The fertility of the mutants was found to be fully fertile in the pollen and spikelets of these mutants ( image 3 B).

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PUM

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Abstract

The invention discloses a method for creating a hybrid affinity line between Asian cultivated rice and African cultivated rice based on gene editing technology and its application in rice distant hybrid vigor. That is, for rice interspecific hybrid sterility S1 African rice candidate genes OgTPR1 Select appropriate gene editing sites and use CRISPR / Cas9 and other site-directed editing technologies to OgTPR1 Perform site-specific knockout. Demonstrating knockout mutant genes ogtpr1 Without affecting the fertility of parent pollen and female gametes, ogtpr1 Hybrid F hybrid between mutant line and Asian cultivated rice 1 The pollen and female gametes have normal fertility, which eliminates the genetic effect of interspecific hybrid sterility. The technical method provided by the present invention can effectively break S1 Locus-mediated reproductive disorders in interspecific hybrids have important application value in utilizing distant hybrid advantages between rice species to increase grain yield.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering technology and crop genetic breeding technology, and specifically relates to a method for creating an interspecific hybrid affinity line between Asian cultivated rice and African cultivated rice and its application in the utilization of rice distant heterosis. Background technique [0002] The world is facing the contradiction of increasing population and decreasing arable land. Using crop heterosis to increase crop yield is of great significance to resolve the food crisis. The genetic relationship between the two parents is more distant, that is, the genetic difference between the two parents is greater, and the performance of hybrid offspring is more obvious, which is called heterosis. However, distant hybrids are usually accompanied by severe hybrid sterility, which greatly hinders the application of crop heterosis. Hybrid sterility is a common phenomenon of interspecies or subspecies ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/46
CPCC12N9/22C12N15/8213C12N15/8287C07K14/415
Inventor 陈乐天刘耀光谢勇尧黄健乐
Owner SOUTH CHINA AGRI UNIV
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