RISE Method Based on Rare Allele Infusion and Sanger Sequencing Estimation: A Simple, Cheap, and Efficient Method for Detecting Transgene Copy Number in Rice

doi:10.1016/j.rsci.2024.05.001

Abstract

Liu Tingchang, Huang Lifang, Liu Peng, Cui Yanchun, Chen Caiyan, Mao Donghai. RISE Method Based on Rare Allele Infusion and Sanger Sequencing Estimation: A Simple, Cheap, and Efficient Method for Detecting Transgene Copy Number in Rice[J]. Rice Science, 2024, 31(5): 499-502.

Figures/Tables 1

Fig. 1. Design and validation of Rare Allele Infusion and Sanger Sequencing Estimation (RISE) method for detecting the copy number of transgenic rice. A, Sequencing peaks of mixtures using different volumes of DNA from two rice varieties (Genit and TB309) with different alleles. The DNA ratios in M1-M5 are shown in Table S3. B, PCR detection of Genit offspring transformed with the PANDA gene, including the TB309 allele. M, DL2000 marker; -, Blank control; +, pC3301-PANDA; L1-L7, Offspring of Genit. C, Phenotype of Genit offspring (L1-L7) transformed with the PANDA gene, including the TB309 allele, treated with 1 mg/L glufosinate. D, Sequencing peaks of Genit offspring (L1-L7) transformed with the PANDA gene, including the TB309 allele. E, Southern blot validation of the copy number in Genit offspring (L1-L7) transformed with the PANDA gene, including the TB309 allele. +, pC3301-PANDA. F, Flowchart depicting the RISE method for detecting copy number in transgenic plants. The single nucleotide polymorphism (SNP) in the target gene or the target gene fused with a tag containing an SNP different from the receptor in the T-DNA of the plasmid is used to transform the recipient variety. The DNA of the transgenic offspring is extracted, and the primer is designed according to the SNP site for Sanger sequencing. The sequencing peak map is analyzed using Sequencher 5.4.5, and the area of the SNP sequencing peak map of the receptor variety is taken as the base number. The copy number of the foreign gene is calculated by the ratio of the SNP base peak area of the tag to the recipient variety. LB, Left border; RB, Right border.

References 31

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