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

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

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 inducing selective RNA silencing.

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