10. Molecular comparison of transgenic plants: Genome and transcriptome approaches

and these are changes that occur following genetic modifications which are aimed to take place as a result of the introduction of the transgene and will consequently result in the accomplishment of the original objective of the genetic engineering process [32]. Unintended effects are those changes that occur following genetic engineering where significant differences are found in the response, phenotype and composition of the GM plant when compared with the tradi-

Unintended effects have further been divided into 'predicable' and 'unpredictable' unintended effects [32]. Predictable unintended effects are changes that exceed the primary expected effects of the introduction of the transgene, but are, however, applicable through the aid of the current knowledge of plant biology and metabolic pathways. On the other hand, unpredictable unintended effects are changes that are currently undefined and not clearly understood. Methods that can be exploited to determine the presence of unintended outcomes of transformation include, among others, determining the transgene integration site/s, the events that occur during the integration of the transgene into the host plant, as well as gene expression analysis of the transgenic genome compared to the traditional counterpart, thus

Guidelines have been set for the molecular characterization of GM crops prior to market and commercial release and these were placed in six categories [53]. These categories are (i) description of the genetic material used for the transformation, including the origin of the donor organism and how the gene was isolated, (ii) description of the transformation method, (iii) description of the transgene loci, (iv) transcript and protein characterization, (v) inheritance and stability of the transgene and (vi) detection and identification of the transgene.

The specified requirements under (i) include information on the plasmid used in the production of the recombinant, detailing genetic elements such as the orientation and position of the transgene expression cassette within the vector, the restriction endonuclease sites of the transformation construct and clearly marked T-DNA borders and promoters. In order to comply, the number of insertion events of the transgene must also be supplied, as well as the transgene insertion site(s). Insertion site detection is expected to be presented as the transgene sequence accompanied by approximately 500 bp of plant DNA in both flanking regions. Possible novel chimeric open reading frames (ORF) should be described and their functionality evaluated. If the flanking sequence contains part of the chimeric ORF, it is expected that more sequencing must be performed beyond the 500 bp radius until a putative

Requirements regarding the expression of the transgene entail, among others, details on the translation of the transcript to protein, tissue specificity of the transcript and protein expression, as well as levels of expression. Furthermore, information on the biochemical, molecular and physiological properties of the transgene product is required as well as the stability of the

protein(s) in the cell and in the surrounding environment.

showing the impact of transformation on the expression of endogenous genes.

9. The need for molecular characterization of GM crops

tional plant from which it is derived.

122 Transgenic Crops - Emerging Trends and Future Perspectives

ORF is obtained.

Several molecular marker techniques that have successfully been used for various research applications, such as cultivar identification, identification of genes for important agricultural traits and marker-assisted selection, can also be applied toward transgenic crops [55]. Molecular marker technologies may therefore serve as rapid and cost-effective methods for genome comparison and as such may be used as an initial screen of recombinant plants.

Simple Sequence Repeats (SSRs), also known as microsatellites, are tandem short oligonucleotide repeat sequences flanked by conserved DNA sequences that can be used to obtain a DNAbased fingerprint of the plant under investigation and are reliable and efficient [56, 57]. Microsatellites are regarded as advantageous as they are simple to perform, low amounts of DNA are required, highly reproducible and the ability to detect high levels of polymorphism [56]. A related marker technique that has been introduced in transgenic crop research is retrotransposon-based markers. The novelty of this technique stems from its ability to reveal extensive chromosomal distribution, as well as randomized genome distribution [58, 59]. Random Amplified Polymorphic DNA (RAPD) techniques are suitable for studies focused on the identification of specific and desired traits and the identification of clonal variants [56], while mutations, insertions and deletions to specific chromosomes or chromosomal regions can be studied through the Restriction Fragment Length Polymorphism (RFLP) technique [60]. For the determination of the insertion site of a transgene and filler DNA, gene-walking methods from known into unknown sequences can be applied [61].

An older technique for gene expression analysis was Northern (mRNA) blotting that only allowed the analysis of a single gene per study. However, developments have facilitated analysis of differential gene expression, or transcript profiling, where the expression of a multitude of genes can be simultaneously analyzed. Differential gene expression has been divided into two categories, namely closed and open architecture systems [62]. A closed system is one where the genes of interest are known and the genome from which the genes are derived has been well characterized [62]. On the other hand, open systems are those that do not require prior knowledge of the transcriptome, as well as the genome of origin.

Several methods, alone or in combination, might be appropriate for optimal gene expression profiling in transgenic plants. Some examples include (not exclusively): Serial Analysis of Gene Expression (SAGE), a gene expression method which allows for quantification and analysis of genes with unknown sequences [63]. This method employs two processes which entail the production of short sequence tags (STTs) from cDNA followed by linking and cloning of these tags for sequencing. LongSAGE enables transcriptome analysis of increased lengths which in turn improves the accuracy of annotating genes [64].

amplification, where two cDNA populations are hybridized to analyze genes that are differentially expressed under set and differing conditions [61]. The obtained difference products are sequenced and analyzed to determine the difference in gene expression levels between the two

Molecular Approaches to Address Intended and Unintended Effects and Substantial Equivalence of Genetically…

http://dx.doi.org/10.5772/intechopen.80339

125

Amplified Fragment Length Polymorphism (AFLP) is a PCR-based technique that has been widely used for its advantages since it utilizes PCR analysis on a small amount of DNA for the identification of various polymorphisms [61]. Several applications have been reported for AFLPs and these include identification of the relatedness of cultivars [56] and the relatedness between transgenic offspring and parental plants [61]. Moreover, the use of mRNA expression analysis through cDNA-AFLPs allows for the evaluation of a large pool of genes differentially expressed between the transgenic and the traditional counterpart. Since it affords the researcher the ability to target coding regions, it facilitates gene expression analysis that leads

Once candidate genes have been identified, qRT-PCR is generally used for quantitative gene expression analysis [76]. This sensitive, highly specific and broad range technique offers researchers the ability to investigate rare transcripts, as well as to analyze multigene families. qRT-PCR is also the technique of choice to measure and quantify expression levels of the inserted transgene(s). However, researchers can only benefit from the effectiveness of this technique if proper internal controls are included. These controls, also known as reference genes, normalize the expression analysis, since they are consistently expressed in tissues of

Due to the non-selective nature of traditional methods of genetic modification, the possibility exists that endogenous genes and their functions will be disrupted through the random integration of the transgene into the plant genome. This phenomenon is linked to unintended effects of genetic modification. Gene expression analysis is thus a crucial part of investigations into the effect of transgene insertion on endogenous gene expression. An understanding of the dynamics of the various available techniques is thus important in selecting the most appropriate technique(s) for the realization of the set objectives. Each method described above has its advantages and limitations. Furthermore, the choice of technique would depend on whether prior knowledge of the host genome is available or not. Using more than one technique in complement would ensure optimum results for investigating comparative / differential gene

This work was supported by the South African Agricultural Research Council (ARC), Department of Science and Technology (DST), National Research Foundation (NRF), Potato South

genomes. A noted disadvantage of this technique is the high levels of labor it requires.

to the identification of genes involved in different biological processes [61, 75].

interest under varying experimental treatments [77].

expression analysis in transgenic crops.

Africa (PSA) and the University of Johannesburg, South Africa.

Acknowledgements

11. Conclusion

Microarrays provide a global view of gene expression and are found in two forms; DNAfragment-based and oligonucleotide-based microarrays [65] with the source of array fragments being either anonymous genomic clones, EST clones or ORF amplified DNA fragments. The advantage of this technique is that a range of both weak and strong signals can be monitored on the same microarray, enabling the simultaneous analysis of a large number of genes. In addition, the technique allows for a pair-wise comparison of samples [66]. However, a major disadvantage of this technique is that an accurate sequence database must be available to facilitate the construction of the microarrays, as well as a large amount of mRNA as starting material to perform the gene expression analysis [65].

With the advent of next-generation sequencing, RNA sequencing (RNASeq or whole transcriptome shotgun sequencing), was developed. RNA-Seq is used to analyze changes in the different RNA species comprising the cellular transcriptome and can inform on the presence and quantity of RNAs in plant samples [67]. Specifically, RNA-Seq facilitates the ability to look at genetic alterations, mutations and changes in gene expression, or differences in gene expression in different groups or treatments such as transgenic – vs. conventional plants.

However, all of the above techniques require substantial amount of sequence information of the genome under investigation. Moreover, availability of funding is another factor for consideration. As a result, alternative gene expression techniques can also be investigated for suitability of intended use [61]. These include mRNA Differential Display (DD), Representational Difference Analysis (RDA), Amplified Fragment Length Polymorphism (AFLP) and quantitative reverse transcriptase real time PCR (qRT-PCR).

Differential gene expression analysis was first performed using mRNA Differential Display (DD) [68]. During DD, cDNA is synthesized from mRNA of each sample of interest, followed by amplification using a combination of anchored oligo-dT and random oligonucleotides. The obtained differentially amplified fragments each represent a transcript or an expressed sequence tag (EST). The advantage of this technique is that it requires a small amount of bioinformatics application during data analysis. Improvements of the technique generated the second generation annealing control primer (ACP)-differential display RT-PCR [69]. Under optimal conditions of use, mRNA DD is a relatively inexpensive but powerful tool, used to identify and isolate differentially expressed transcripts, as well as for comparative studies between several mRNA populations [70].

Subtractive hybridization of mRNA is another method that has been employed to differentially identify mRNAs associated with a cell- or tissue type or cellular responses [71]. A reduction in the number of genes in need of analysis in a comparative transgenic study is an important advantage [72]. Another advantage of the technique is its ability to reveal lower abundance transcripts [73], but the technique is also time consuming and labor intensive.

Representational Difference Analysis (RDA) is a subtractive DNA enrichment technique designed to identify differences between two genomes without quantifying expression levels [74]. The technique was later modified by using cDNA as template to facilitate the detection of rare transcripts. cDNA-RDA utilizes subtractive DNA enrichment in association with PCR amplification, where two cDNA populations are hybridized to analyze genes that are differentially expressed under set and differing conditions [61]. The obtained difference products are sequenced and analyzed to determine the difference in gene expression levels between the two genomes. A noted disadvantage of this technique is the high levels of labor it requires.

Amplified Fragment Length Polymorphism (AFLP) is a PCR-based technique that has been widely used for its advantages since it utilizes PCR analysis on a small amount of DNA for the identification of various polymorphisms [61]. Several applications have been reported for AFLPs and these include identification of the relatedness of cultivars [56] and the relatedness between transgenic offspring and parental plants [61]. Moreover, the use of mRNA expression analysis through cDNA-AFLPs allows for the evaluation of a large pool of genes differentially expressed between the transgenic and the traditional counterpart. Since it affords the researcher the ability to target coding regions, it facilitates gene expression analysis that leads to the identification of genes involved in different biological processes [61, 75].

Once candidate genes have been identified, qRT-PCR is generally used for quantitative gene expression analysis [76]. This sensitive, highly specific and broad range technique offers researchers the ability to investigate rare transcripts, as well as to analyze multigene families. qRT-PCR is also the technique of choice to measure and quantify expression levels of the inserted transgene(s). However, researchers can only benefit from the effectiveness of this technique if proper internal controls are included. These controls, also known as reference genes, normalize the expression analysis, since they are consistently expressed in tissues of interest under varying experimental treatments [77].
