Gametocidal(Gc)chromosome can kill the gametes without themselves by causing chromosomal breakage to ensure their preferential transmission,that is Gc genes have the dual functions,one of which gives “breaking” function and another “protecting” function.As the Gc chromosome can induce chromosomal breakage and chromosomal recombination,the Gc chromosome can be used as an effective tool to genetic breeding.The Gc system can be used to generate the deletion and translocation lines in order to transfer the beneficial genes to wheat,however,the molecular mechanism of Gc action remains unclear.MiRNAs are small,endogenous and noncoding RNAs which have crucial regulatory roles and can participate in DNA methylation,male sterile,DNA damage response,chromosomal breaks,cell division and cell death.The 24-nt siRNAs can play important roles in the genome reconstruction and stabilization.In order to investigate whether these two categories of sRNAs are related to the Gc action,two small RNA libraries from Triticum aestivum L.Chinese Spring(CS)and Chinese Spring-gametocidal 3C chromosome monosomic addition line(CS-3C)were constructed and sequenced.Base on sequencing data,we identified 123 conserved miRNAs and 438 putative novel miRNAs.The differential expression analysis and TaqMan qRT-PCR revealed that 48 conserved miRNAs and 3 putative novel miRNAs were differentially expressed between these two plant materials.In our study,some miRNAs whose target genes were related to the fertility or chromosomal breakage were dramatically up-regulated in CS-3C,such as miR159a and miR 9657b-3p.As reported that tae-miR159 can participate in the development of anthers and the accumulation of tae-miR159 in transgenic rice can result in male sterility.Thus,miRNAs like miR159a and miR9657b-3p which up-regulated in CS-3C may be considered as “breaking” miRNAs which maybe participate in the “break” function in Gc action while miRNAs which down-regulated in CS-3C,like miR167d-5p and miR167e-5p,may be considered as “protecting” miRNAs.For siRNAs,the distribution trend of siRNAs were conducted and the result indicated that both in CS and CS-3C,the siRNAs with highest peak densities distributed near the 1kb upstream and downstream of the transcribed regions.In order to verify the relationship between 24nt siRNAs and the DNA methylation,we selected the LINE/L1 which corresponded to the siRNAs which were dramatically down-regulated in CS-3C to conduct the bisulfite sequencing analysis.The bisulfite sequencing results indicated that the LINE/L1 retrotransposons showed a generally reduced CHH methylation(hypo-mCHH)in the CS-3C plants as the corresponding siRNAs was reduced in CS-3C.Such results maybe because the CHH methylation in this region were generated de novo by the 24-nt siRNA-directed RdDM pathway.The 24-nt siRNAs maybe mainly participate in the “breaking” function of Gametocidal action by down-regulating the DNA methylation,mainly the CHH methylation and activation of TEs.Our data offered evidences that sRNAs(miRNAs and siRNAs)maybe play important roles in the Gametocidal action mainly the “breaking” function.