**6. What do phenotypic changes induced by RNA silencing in soybean indicate?**

Soybean is thought to be derived from an ancestral plant(s) with a tetraploid genome, and as a consequence, large portions of the soybean genome are duplicated [7], with nearly 75% of the genes present in multiple copies [117]. In addition, genes in the soybean genome are sometimes duplicated in tandem [118-121]. Our recent studies have indeed shown function‐ al redundancy of duplicated genes in soybean [122, 123]. Such gene duplication can be an obstacle to producing mutants by conventional methods of mutagenesis. In this regard, the gene silencing technique is particularly useful because it allows silencing of multiple cog‐ nate genes having nucleotide sequence identity.

Changes in phenotypes as a consequence of inducing RNA silencing have been successful for many genes in soybean as mentioned above. Considering that many genes are duplicat‐ ed in soybean genome, this fact indicates either that RNA silencing worked on all duplicated genes that have the same function or that the genes were subfunctionalized after duplica‐ tion, so that RNA silencing of even a single gene of the duplicated genes resulted in the phe‐ notypic changes.

It is of interest to understand whether duplicated genes have identical or diversified func‐ tions, which may depend on the time after duplication event and/or the selection pressure on the genes. To analyze the functions of each copy of the duplicated genes, we need to si‐ lence a specific copy of the duplicated genes. If the duplicated genes are expressed in differ‐ ent tissues, RNA silencing of both genes can lead to understanding the function of each gene. PTGS by transcribing inverted repeat with a constitutive promoter or VIGS will be suitable for this analysis. An example of such an approach is the VIGS of duplicated *TFL1* orthologues, which are expressed in different tissues. A specific role of one of the *TFL1* or‐ thologues has been identified by VIGS as mentioned earlier [102].
