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

摘要/Abstract

. [J]. Rice Science, 2024, 31(5): 499-502.

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.

参考文献 31

[1]	Addison C K, Angira B, Cerioli T, Groth D E, Richards J K, Linscombe S D, Famoso A N. 2021. Identification and mapping of a novel resistance gene to the rice pathogen, Cercospora janseana. Theor Appl Genet, 134(7): 2221-2234.
[2]	Adjah K L, Asante M D, Toure A, Aziadekey M, Amoako-Andoh F O, Frei M, Diallo Y, Agboka K. 2022. Improvement of rice production under drought conditions in West Africa: Application of QTLs in breeding for drought resistance. Rice Sci, 29(6): 512-521.
[3]	Avni R, Oren L, Shabtay G, Assili S, Pozniak C, Hale I, Ben-David R, Peleg Z, Distelfeld A. 2018. Genome based meta-QTL analysis of grain weight in tetraploid wheat identifies rare alleles of GRF4 associated with larger grains. Genes, 9(12): 636.
[4]	Cai Y M, Dudley Q M, Patron N J. 2021. Measurement of transgene copy number in plants using droplet digital PCR. Bio-protocol, 11(13): e4075.
[5]	Chen J, Gao H, Zheng X M, Jin M N, Weng J F, Ma J, Ren Y L, Zhou K N, Wang Q, Wang J, Wang J L, Zhang X, Cheng Z J, Wu C Y, Wang H Y, Wan J M. 2015. An evolutionarily conserved gene, FUWA, plays a role in determining panicle architecture, grain shape and grain weight in rice. Plant J, 83(3): 427-438.
[6]	de Saeger J, Park J, Thoris K, de Bruyn C, Chung H S, Inzé D, Depuydt S. 2022. IMPLANT: A new technique for transgene copy number estimation in plants using a single end-point PCR reaction. Plant Methods, 18(1): 132.
[7]	Famoso A N, Zhao K Y, Clark R T, Tung C W, Wright M H, Bustamante C, Kochian L V, McCouch S R. 2011. Genetic architecture of aluminum tolerance in rice (Oryza sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genet, 7(8): e1002221.
[8]	Fan F F, Liu M M, Li N N, Guo Y, Yuan H R, Si F F, Cheng M X, Chen G L, Cai M, Li N W, Zhang Y X, Yu Y, Pi L M, Yang H C, Yang F, Wang K, Li S Q. 2023. Gain-of-function allele of HPY1 coordinates source and sink to increase grain yield in rice. Sci Bull, 68(19): 2155-2159.
[9]	Gross B L, Skare K J, Olsen K M. 2009. Novel Phr1 mutations and the evolution of phenol reaction variation in US weedy rice (Oryza sativa). New Phytol, 184(4): 842-850.
[10]	Hu J, Wang Y X, Fang Y X, Zeng L J, Xu J, Yu H P, Shi Z Y, Pan J J, Zhang D, Kang S J, Zhu L, Dong G J, Guo L B, Zeng D L, Zhang G H, Xie L H, Xiong G S, Li J Y, Qian Q. 2015. A rare allele of GS2 enhances grain size and grain yield in rice. Mol Plant, 8(10): 1455-1465.
[11]	Jagadeesh D, Prasanna Kumar M K, Chandrakanth R, Devaki N S. 2018. Molecular diversity of internal transcribed spacer among the monoconidial isolates of Magnaporthe oryzae isolated from rice in Southern Karnataka, India. J Genet Eng Biotechnol, 16(2): 631-638. DOI PMID
[12]	Jan A, Kitano H, Matsumoto H, Komatsu S. 2006. The rice OsGAE1 is a novel gibberellin-regulated gene and involved in rice growth. Plant Mol Biol, 62(3): 439-452.
[13]	Li H, Wu C Y, Du M M, Chen Y C, Hou X, Yang Y N, Xie K B. 2021. A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants. Mol Breed, 41(2): 13.
[14]	Long Y M, Chao W S, Ma G J, Xu S S, Qi L L. 2017. An innovative SNP genotyping method adapting to multiple platforms and throughputs. Theor Appl Genet, 130(3): 597-607. DOI PMID
[15]	Mao D H, Tao S T, Li X, Gao D Y, Tang M F, Liu C B, Wu D, Bai L L, He Z K, Wang X D, Yang L, Zhu Y X, Zhang D C, Zhang W L, Chen C Y. 2022. The Harbinger transposon-derived gene PANDA epigenetically coordinates panicle number and grain size in rice. Plant Biotechnol J, 20(6): 1154-1166.
[16]	Marmiroli N, Maestri E, Gullì M, Malcevschi A, Peano C, Bordoni R, de Bellis G. 2008. Methods for detection of GMOs in food and feed. Anal Bioanal Chem, 392(3): 369-384. DOI PMID
[17]	Masterson J. 1994. Stomatal size in fossil plants: Evidence for polyploidy in majority of angiosperms. Science, 264: 421-424. DOI PMID
[18]	Nitcher R, Pearce S, Tranquilli G, Zhang X Q, Dubcovsky J. 2014. Effect of the hope FT-B1 allele on wheat heading time and yield components. J Hered, 105(5): 666-675.
[19]	Scott M P. 2009. Transgenic Maize:Methods and Protocols. New Jersey, USA: Humana Press.
[20]	Shen J, Kirk B D, Ma J P, Wang Q H. 2009. Diversifying selective pressure on influenza B virus hemagglutinin. J Med Virol, 81(1): 114-124. DOI PMID
[21]	Song X J, Kuroha T, Ayano M, Furuta T, Nagai K, Komeda N, Segami S, Miura K, Ogawa D, Kamura T, Suzuki T, Higashiyama T, Yamasaki M, Mori H, Inukai Y, Wu J Z, Kitano H, Sakakibara H, Jacobsen S E, Ashikari M. 2015. Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice. Proc Natl Acad Sci USA, 112(1): 76-81. DOI PMID
[22]	Xu Y, Chen B, He Q H, Qiu Y L, Liu X, He Z Y, Xiong Z P. 2014. New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B₁ based on using peptide-MBP fusion protein as substitute for coating antigen. Anal Chem, 86(16): 8433-8440.
[23]	Xue D Y. 2009. Safety and Management of Genetically Modified Organisms. Beijing, China: Science Press. (in Chinese)
[24]	Yang L T, Ding J Y, Zhang C M, Jia J W, Weng H B, Liu W X, Zhang D B. 2005. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep, 23(10/11): 759-763.
[25]	Young S A, White F F, Hopkins C M, Leach J E. 1994. AVRXa10 protein is in the cytoplasm of Xanthomonas oryzae pv. oryzae. Mol Plant Microbe Interact, 7(6): 799-804.
[26]	Yu S, Xia D X, Luo Q X, Cheng Y X, Takano T, Liu S K. 2007. Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli. Protein Expr Purif, 52(2): 379-383.
[27]	Zhang L, Wang R C, Xing Y D, Xu Y F, Xiong D P, Wang Y M, Yao S G. 2021. Separable regulation of POW1 in grain size and leaf angle development in rice. Plant Biotechnol J, 19(12): 2517-2531. DOI PMID
[28]	Zhang N, Zhang L N, Tao Y, Guo L, Sun J, Li X, Zhao N, Peng J, Li X J, Zeng L, Chen J S, Yang G P. 2015. Construction of a high density SNP linkage map of kelp (Saccharina japonica) by sequencing TaqI site associated DNA and mapping of a sex determining locus. BMC Genomics, 16(1): 189.
[29]	Zhang X J, Wang J F, Huang J, Lan H X, Wang C L, Yin C F, Wu Y Y, Tang H J, Qian Q, Li J Y, Zhang H S. 2012. Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice. Proc Natl Acad Sci USA, 109: 21534-21539.
[30]	Zhang Y D, Zhu Z, Zhao Q Y, Chen T, Yao S, Zhou L H, Zhao L, Zhao C F, Wang C L. 2016. Haplotypes of qGL3 and their roles in grain size regulation with GS3 alleles in rice. Genet Mol Res, 15(1): gmr.15017587.
[31]	Zhou C, Liu X Y, Li X L, Zhou H L, Wang S J, Yuan Z, Zhang Y H, Li S H, You A Q, Zhou L, He Z Q. 2021. A genome doubling event reshapes rice morphology and products by modulating chromatin signatures and gene expression profiling. Rice, 14(1): 72.

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