A non-PCR-based Invader® assay quantitatively detects single-copy genes in complex plant genomes

Gupta, Manju; Nirunsuksiri, Wilas; Schulenberg, Greg; Hartl, Thomas; Novak, Stephen J.; Bryan, Jill; VanOpdorp, Nathan; Bing, J. W.; Thompson, S.W.N.

doi:10.1007/s11032-007-9117-7

Cited by 6 publications

(2 citation statements)

References 17 publications

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“…Although Southern blots can provide highly accurate measures of copy number when multiple probes and digests are used, the procedures are lengthy and laborious. The Invader or Taqman assays provide a high throughput analytical tool for copy number assessment for large number of transgenic plants with similar data quality (Pielberg et al 2003;Bubner and Baldwin 2004;Gupta et al 2008).…”

Section: Discussionmentioning

confidence: 99%

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

Williams

Shen

et al. 2010

Transgenic Res

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Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.

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Section: Discussionmentioning

confidence: 99%

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

Williams

Shen

et al. 2010

Transgenic Res

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“…The use of hairy roots to validate promoter activity is a simple alternative for gauging promoter strength in stably-transformed plants, although the influence of copy number on gene expression should be considered [ 69 ]. The transient expression analysis used in this research may nevertheless be more reflective of general promoter strength as each cell receives similar high copy numbers of each DNA construct, and hundreds to thousands of cells are collectively analyzed.…”

Section: Discussionmentioning

confidence: 99%

High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

et al. 2010

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BackgroundAlthough numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max).ResultsIn this research, a bioinformatics approach was first performed to identify members of the Gmubi (G.max ubiquitin) and the GmERF (G. max Ethylene Response Factor) gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus) cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression.ConclusionIn this study, we present expression intensity data on 20 novel soybean promoters from two different gene families, ubiquitin and ERF. We also demonstrate the utility of lima bean cotyledons and soybean hairy roots for rapid promoter analyses and provide novel insights towards the utilization of these expression systems. The soybean promoters characterized here will be useful for production of transgenic soybean plants for both basic research and commercial plant improvement.

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New Technologies for Ultra-High Throughput Genotyping in Plants

Appleby

Edwards²,

Batley

2009

Methods in Molecular Biology™

123

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Molecular genetic markers represent one of the most powerful tools for the analysis of plant genomes and the association of heritable traits with underlying genetic variation. Molecular marker technology has developed rapidly over the last decade, with the development of high-throughput genotyping methods. Two forms of sequence-based marker, simple sequence repeats (SSRs), also known as microsatellites and single nucleotide polymorphisms (SNPs) now predominate applications in modern plant genetic analysis, along the anonymous marker systems such as amplified fragment length polymorphisms (AFLPs) and diversity array technology (DArT). The reducing cost of DNA sequencing and increasing availability of large sequence data sets permits the mining of this data for large numbers of SSRs and SNPs. These may then be used in applications such as genetic linkage analysis and trait mapping, diversity analysis, association studies and marker-assisted selection. Here, we describe automated methods for the discovery of molecular markers and new technologies for high-throughput, low-cost molecular marker genotyping. Genotyping examples include multiplexing of SSRs using Multiplex-Ready marker technology (MRT); DArT genotyping; SNP genotyping using the Invader assay, the single base extension (SBE), oligonucleotide ligation assay (OLA) SNPlex system, and Illumina GoldenGate and Infinium methods.

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A non-PCR-based Invader® assay quantitatively detects single-copy genes in complex plant genomes

Cited by 6 publications

References 17 publications

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

New Technologies for Ultra-High Throughput Genotyping in Plants

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