**9. Conclusion and perspectives**

RNA silencing has been used as a powerful tool to engineer novel traits or analyze gene function in soybean. Soybean plants that have engineered a metabolic pathway or acquired resistance to diseases have been produced by transgene-induced gene silencing. VIGS has been used as a tool to analyze gene function in soybean. In addition to RNA silencing, sitedirected mutagenesis using zinc-finger nucleases has been applied to mutagenizing dupli‐ cated genes in soybean [138]. Such reverse genetic approaches may be supplemented by forward genetic approaches such as high linear energy transfer radiation-based mutagene‐ sis, e.g., irradiation of ion beam [139] and fast neutron [140]. Similarly, gene tagging systems using maize Ds transposon [141] and rice *mPing* transposon [142] have also been developed in soybean. Aside from using RNA silencing as a tool to engineer novel traits, analysis of mutants in combination with reverse genetic approaches may facilitate the identification of causative gene(s) of the mutation. An interesting feature of RNA silencing is its inducible nature, which allows downregulation of a gene in a tissue-specific manner. This strategy is particularly advantageous for analyzing the function of genes whose mutation or ubiquitous downregulation is lethal. Another feature of RNA silencing is that it allows analysis of bio‐ logical phenomena that involve the effect of a difference in the mRNA level of the gene. The dependence of pigmentation in soybean pubescence on the mRNA level of the *F3′H* gene has actually been shown by utilizing VIGS [143]. In this regard, selective RNA silencing of duplicated genes may reveal the presence of additive effects of the expression levels of du‐ plicated genes in soybean.

### *Abbreviations*

**8. Differentiation of duplicated genes and induction of RNA silencing**

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

though the efficiency of silencing may depend on the system of silencing induction.

inducing selective RNA silencing.

Relationships

518

**9. Conclusion and perspectives**

genes, which may have a deleterious effect on the organism.

How much sequence difference will be necessary to induce selective RNA silencing? A fac‐ tor that affects induction of RNA silencing is the extent of sequence identity between the dsRNA that triggers RNA silencing and its target gene. IR-PTGS could be induced by IRtranscripts that can form 98-nt or longer dsRNAs [39]. In VIGS, the lower size limit of the inserted fragments required for inducing PTGS is 23-nt, a size almost corresponding to that of siRNAs [134], and that for inducing TGS is 81-91 nt [135]. Silencing a gene probably re‐ quires sequence identity longer than the size of siRNAs between dsRNA and its target, al‐

We previously induced *CHS* VIGS in soybean [56]. In soybean seed coats, the *CHS7/CHS8* genes, which share 98% nucleotide sequence identity in the coding region, are predominant‐ ly expressed among the eight members of the *CHS* gene family [136, 137]. We have induced the silencing using a virus vector that carried a 244-nt fragment of the *CHS7* gene [56]. The *CHS* mRNA levels in the seed coats and leaf tissues of plants infected with the virus were reduced to 12.4% and 47.0% of the control plants, respectively. One plausible explanation for the differential effects of VIGS on these tissues may be that the limited sequence homology (79%-80%) between the *CHS7* and the *CHS1-CHS3* genes, the transcripts of which make up approximately 40% of the total *CHS* transcript content of leaf tissues [137], results in the deg‐ radation of the *CHS1-CHS3* transcripts at a lower efficiency than the degradation of *CHS7/ CHS8* transcripts. Consistent with these results, naturally occurring *CHS* RNA silencing, in which *CHS7/CHS8* genes are silenced in seed coat tissues, is thought to be induced by in‐ verted repeat transcripts of a *CHS3* gene segment [110]. In terms of the practical use of trans‐ gene-induced RNA silencing, these results suggest that a portion of genes whose sequence identity between duplicated genes is lower than 79%-80% should be chosen as a target for

The naturally occurring RNA silencing of the *CHS* genes in soybean may indicate relation‐ ships between diversification of duplicated genes and RNA silencing. Gene duplication can be a cause of RNA silencing because it may sometimes result in the production of dsRNA, which triggers RNA silencing through read-through transcription [114, 115]. In the *CHS* si‐ lencing in soybean, the extent of mRNA decrease differs between different copies of the gene family. These observations may indicate that plants use subfunctionalization of dupli‐ cated genes as a means to avoid the occurrence of simultaneous silencing of duplicated

RNA silencing has been used as a powerful tool to engineer novel traits or analyze gene function in soybean. Soybean plants that have engineered a metabolic pathway or acquired resistance to diseases have been produced by transgene-induced gene silencing. VIGS has been used as a tool to analyze gene function in soybean. In addition to RNA silencing, siteAGO, Argonaute; ALSV, *Apple latent spherical virus*; amiRNA, artificial miRNA; BPMV, *Bean pod mottle virus*; CaMV, *Cauliflower mosaic virus*; CHS, chalcone synthase; CMV, *Cucumber mosaic virus*; dsRNA, double-stranded RNA; EST, expressed sequence tag; F3′H, flavonoid 3′-hydroxylase; IR, inverted repeat; miRNA, micro RNA; MYA, million years ago; PTGS, post-transcriptional gene silencing; RdRP, RNA-dependent RNA polymerase; RISC, RNAinduced silencing complex; RNAi, RNA interference; siRNA, short interfering RNA; SMV, *Soybean mosaic virus*; TFL1, TERMINAL FLOWER1; TGS, transcriptional gene silencing; UTR, untanslated region; VIGS, virus-induced gene silencing
