Recombined nucleic acid fragment reccr020325 and its detection method
A technology for recombining nucleic acid and fragments, which is applied in the field of recombinant nucleic acid fragments and its detection, which can solve problems such as increased production costs, increased resistance of brown planthoppers, and environmental pollution, and achieve the effect of improved resistance
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Embodiment 1
[0042] Example 1 Breeding of Recombinant Plants Introduced with Resistant Genome Fragment of Brown Planthopper
[0043] The materials used in this example are rice 'Yuehui 9113' and rice 'Hua 3418B'.
[0044] Rice 'Hua 3418B' has good resistance to brown planthopper, and it is speculated that it may be Bph3 (Liu et al., Nature Biotechnology.2015,33:301-305), QBph4 (Hu et al., Molecular Breeding.2015,35:3) of chromosome 4 ) and Bph15 (Yang et al., Theoretical and Applied Genetics. 2004, 110: 182-191) gene cluster region played a key role in the material's resistance to brown planthopper.
[0045] 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. Some of the molecular markers used were from the website http: / / www.gramene.org / , and some were designed by ourselves. The design method is to download the DNA sequenc...
Embodiment 2
[0055] Example 2 Determination of Homologous Recombination Fragments After Introducing the Resistance Genome Fragments of Brown Planthopper
[0056] In order to determine the size of the imported brown planthopper-resistant genome fragment, homologous recombination fragments on both sides of the target genome fragment were sequenced for the homozygous single plant that introduced the brown planthopper-resistant genome fragment into 'Yuehui 9113'. The N. lugens resistant genome recombination nucleic acid fragment contained in CR020325 was named RecCR020325.
[0057] It was preliminarily determined by the rice genome-wide breeding chip RICE60K detection results that RecCR020325 was located between two SNP markers R0406475020AG and F0406988115GA.
[0058] At the same time, three samples of 'Yuehui 9113', 'Hua 3418B' and CR020325 were sequenced using Miseq sequencing technology. The TruSeq Nano DNA LT Kit (illumina) kit was used for library establishment, the LibraryQuantifica...
Embodiment 3
[0069] Example 3 Identification of resistance to 'Yuehui 9113' after introducing genome fragments resistant to brown planthopper
[0070] In order to identify the brown planthopper resistance of the selected plants containing recombinant nucleic acid fragments, the new line CR020325 bred by the application, the recipient parent 'Yuehui 9113', the donor parent 'Hua 3418B' (as a positive control), and the high-sensitivity The brown planthopper variety 'Taizhong Zailai No. 1' (as a negative control) was tested for indoor resistance to brown planthopper, and the identification method was as follows.
[0071] Soak the seeds and germination of each material in the room, and then sow them in plastic basins with grid lines. Each material is divided into 3 rows and a total of 45 plants are sown. When the two leaves are one heart, 10 plants are kept in each row, and a total of 30 plants grow healthy. Consistent plants are used for inoculation. The source of insects came from brown p...
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