Recombinant nucleic acid fragment RecCR010165 and detection method thereof

A technology for recombining nucleic acids and fragments, applied in the field of recombinant nucleic acid fragments and their detection, can solve the problems of low efficiency and long time, and achieve the effects of improving rice blast resistance and improving rice blast resistance.

Active Publication Date: 2017-10-24
CHINA NAT SEED GRP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002]For a long time, the selection method of traditional breeding has mainly relied on th...

Method used

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  • Recombinant nucleic acid fragment RecCR010165 and detection method thereof
  • Recombinant nucleic acid fragment RecCR010165 and detection method thereof
  • Recombinant nucleic acid fragment RecCR010165 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Breeding of Recombinant Plants Introduced with Blast Resistance Genome Fragment

[0041] The materials used in this example are rice 'Tianfeng B' and rice 'Huafeng 1B'.

[0042] The rice 'Huafeng 1B' has good resistance to rice blast, and it is speculated that the gene cluster region where Pi2, Pi9 and Pigm of chromosome 6 are located may play a key role in the rice blast resistance of this material.

[0043] During the breeding process of the recombinant plants, the molecular markers were used to perform prospect selection on the recombinant plants, and the adopted molecular markers for prospect selection were screened. Referring to the rice Nipponbare genome MSU / TIGR annotation version 6.1, download the DNA sequence of chromosome 6 from 9,559,000 to 10,990,000. The SSR sites in the above sequences were scanned using SSRLocator software. Primer Premier 3.0 software was used to design primers for the found SSR loci, and a total of 162 pairs of primers wer...

Embodiment 2

[0053] Example 2 Determination of Homologous Recombination Fragments After Introducing Blast Resistance Genome Fragments

[0054] In order to determine the size of the imported rice blast resistance genome fragment, the homozygous single plant of the imported fragment of 'Tianfeng B' was sequenced for homologous recombination fragments on both sides of the target genome fragment. The recombinant nucleic acid fragment of the rice blast resistance genome contained in CR010165 was named RecCR010165.

[0055] It was preliminarily determined by the rice genome-wide breeding chip RICE60K detection results that RecCR010165 was located between two SNP markers R0610372586AG and F0610880381TC.

[0056] At the same time, three samples of 'Tianfeng B', 'Huafeng 1B' and CR010165 were subjected to whole-genome sequencing using Miseq sequencing technology. The TruSeq Nano DNA LT Kit (illumina) kit was used for library establishment, the Library Quantification Kit–Universal (KAPA Biosyste...

Embodiment 3

[0066] Example 3 Resistance Identification of 'Tianfeng B' After Introducing Blast Resistance Genome Fragment

[0067] In order to identify the resistance effect, the new line CR010165 selected by the application, the recurrent parent 'Tianfeng B', the rice blast resistant variety Gumei No. 4 (as a positive control), and the rice blast susceptible variety Lijiang Xintuan Heigu ( As a negative control) for indoor planting, it is cultivated to the 3-4 leaf stage and then identified by the following methods:

[0068]Two blast strains, 4105-1 isolated from rice blast samples in Hunan disease nurseries and 1209-1 isolated from rice blast samples in Guangdong disease nurseries in 2013, were selected as inoculation strains. The strain was stored at -20°C by the sorghum grain method. Before use, the preserved sorghum grains were taken out and activated on a potato dextrose medium (PDA) plate (PDA: 200g peeled potatoes, 20g glucose, 15g agar powder, distilled water to 1L), After cu...

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Abstract

The invention provides a recombinant nucleic acid fragment and a detection method thereof. The invention further provides a breeding method of a rice plant containing the recombinant nucleic acid fragment, which performs foreground selection and background selection on a recombinant plant through a molecular marker, so as to obtain the rice plant containing the recombinant nucleic acid fragment.

Description

technical field [0001] This application relates to genome-wide selective breeding technology. Specifically, the present application relates to the selection and breeding of rice plants containing recombinant nucleic acid fragments using genome-wide selective breeding technology, as well as the resulting recombinant nucleic acid fragments and their detection methods. Background technique [0002] For a long time, the selection method of traditional breeding has mainly relied on the evaluation of field phenotypes, making choices based on the breeder's personal experience. The biggest disadvantage is that it takes a long time and is low in efficiency. To improve the efficiency of selection, the most ideal method should be to be able to directly select the genotype. With the development of molecular biotechnology, molecular markers provide the possibility for direct selection of genotypes. In recent years, molecular marker-assisted selection methods have been used to improve i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11A01H1/02A01H1/04
CPCA01H1/02A01H1/04C12Q1/6895C12Q2600/156
Inventor 周发松喻辉辉邱树青江峥何宗顺雷昉姚玥李旭冯芳李菁韦懿陈光牟同敏张启发
Owner CHINA NAT SEED GRP
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