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Soybean, *Glycine max* (L.) Merr., is considered to be a typical paleopolyploid species with a complex genome [1-3]. Approximately 70 to 80% of angiosperm species have undergone polyploidization at some point in their evolutionary history, which is a well-known mecha‐ nism of gene duplication in plants [4]. The soybean genome actually possesses a high level of duplicate sequences, and furthermore, possesses homoeologous duplicated regions, which are scattered across different linkage groups [5-8]. Based on the genetic distances esti‐ mated by synonymous substitution measurements for the pairs of duplicated transcripts from expressed sequence tag (EST) collections of soybean and *Medicago truncatula*, Schlueter et al. estimated that soybean probably underwent two major genome duplication events: one that took place 15 million years ago (MYA) and another 44 MYA [9].

Gene duplication is a major source of evolutionary novelties and can occur through duplica‐ tion of individual genes, chromosomal segments, or entire genomes (polyploidization). Un‐ der the classic model of duplicate gene evolution, one of the duplicated genes is free to accumulate mutations, which results in either the inactivation of transcription and/or a func‐ tion (pseudogenization or nonfunctionalization) or the gain of a new function (neofunction‐ alization) as long as another copy retains the requisite physiological functions [10; and references therein]. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classic model [11].

Recent advances in genome study have led to the formulation of several evolutionary mod‐ els: a model proposed by Hughes [12] suggests that gene sharing, whereby a single gene encodes a protein with two distinct functions, precedes the evolution of two functionally distinct proteins; the duplication–degeneration–complementation model suggests that du‐ plicate genes acquire debilitating yet complementary mutations that alter one or more sub‐

© 2013 Kasai et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kasai et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

functions of the single gene progenitor, an evolutionary consequence for duplicated loci referred to as subfunctionalization [4, 11, 13]. In addition to this notion, models involving epigenetic silencing of duplicate genes [14] or purifying selection for gene balance [15, 16] have also been proposed. In soybean, differential patterns of expression have often been de‐ tected between homoeologous genes [17, 18], which indicates that subfunctionalization has occurred in these genes.

When the extent of subfunctionalization is limited, mutations in only one of multiple cog‐ nate gene copies do not often result in phenotypic changes. Therefore, methods that allow suppression of all copies of the duplicated gene are required for analyzing gene function or engineering novel traits. RNA silencing refers collectively to diverse RNA-mediated path‐ ways of nucleotide-sequence-specific inhibition of gene expression, either at the posttran‐ scriptional or transcriptional level, which provides a powerful tool to downregulate a gene or a gene family [19, 20]. Suppression of gene expression through RNA silencing is particu‐ larly useful for analyzing the function(s) of duplicated genes or engineering novel traits be‐ cause it allows silencing of multiple cognate genes having nucleotide sequence identity. In fact, to produce soybean lines that have a novel trait, researchers have frequently used RNA silencing induced by a transgene.

In this review, we describe application of RNA silencing to understand the roles of genes or engineering novel traits in soybean. We describe methods to induce simultaneous silencing of duplicated genes and selective silencing of each copy of duplicated genes through RNA silencing. In addition to intentionally induced RNA silencing, we also refer to naturally oc‐ curring RNA silencing. Based on our knowledge of RNA silencing in soybean, we propose a hypothesis that plants may have used subfunctionalization of duplicated genes as a means to avoid the occurrence of simultaneous silencing of duplicated genes, which could be dele‐ terious to the organism.
