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Recombinant nucleic acid fragment RecCR012602 and detection method thereof

A technology for recombining nucleic acid and fragments, applied in the field of recombining nucleic acid fragments and their detection, can solve the problems of time-consuming and low efficiency

Active Publication Date: 2017-06-27
CHINA NAT SEED GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[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. Low

Method used

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  • Recombinant nucleic acid fragment RecCR012602 and detection method thereof
  • Recombinant nucleic acid fragment RecCR012602 and detection method thereof
  • Recombinant nucleic acid fragment RecCR012602 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 'HD9802S' and rice 'R6'.

[0042] Rice 'R6' has good blast resistance, 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 were designed. Through the PCR met...

Embodiment 2

[0054] Example 2 Determination of Homologous Recombination Fragments After Introducing Blast Resistance Genome Fragments

[0055] In order to determine the size of the imported rice blast resistance genome fragment, the homozygous individual plants of the 'HD9802S' imported fragment were 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 CR012602 was named RecCR012602.

[0056] It was preliminarily determined by the rice genome-wide breeding chip RICE60K detection results that RecCR012602 was located between two SNP markers R0610084130TG and R0610454932GA.

[0057] At the same time, three samples of 'HD9802S', 'R6' and CR012602 were sequenced whole-genome using Miseq sequencing technology. The TruSeq Nano DNA LT Kit (illumina) kit was used for library establishment, the Library Quantification Kit–Universal (KAPA Biosystems) kit was used for quant...

Embodiment 3

[0068] Example 3 Resistance Identification of Rice Blast Resistance Genomic Fragment Imported into 'HD9802S'

[0069] In order to identify the resistance effect, the new line CR012602 selected by the application, the recurrent parent 'HD9802S', the rice blast disease-resistant variety Gumei No. 4 (as a positive control), and the rice blast disease-susceptible variety Lijiang Xintuan Heigu (as a negative control) Control) is carried out indoor planting, and adopts following method to identify after it is cultivated to the 3-4 leaf stage:

[0070] Select M15Bb-1-1, M15Bb-1-2, M15Bb-2-1, M15Bb-3-1, M15Bb-4-1, M15Bb-5-1 isolated from rice blast disease samples in Yichang disease nursery, Hubei in 2015 and M15Bb-6-1, a total of 7 blast strains were used 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 glu...

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Abstract

The invention provides a recombinant nucleic acid fragment RecCR012602 and a detection method thereof. The invention also provides a method for breeding a rice plant containing the recombinant nucleic acid fragment. A molecular marker is utilized for carrying out foreground selection and background selection on a recombinant plant, and the rice plant containing the recombinant nucleic acid fragment is obtained.

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
CPCA01H1/02A01H1/04C12Q1/6895
Inventor 周发松喻辉辉曹志邱树青张学堂张龙雨雷昉姚玥李旭江峥李菁韦懿何予卿张启发
Owner CHINA NAT SEED GRP
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