Role Of Small RNAs In Gametocidal Action In Wheat

Gametocidal(Gc)chromosomes can kill the gametes without themselves by causing chromosomal breakage and 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 further cause chromosomal recombination,it has been used for creating translocation and deletion lines to transfer the beneficial alien genes to common wheat.However,the molecular mechanism of Gc action remains unclear so far.In this study,small RNA libraries from Triticum aestivum cv.Chinese Spring(CS)and Chinese Spring-Gc 3C chromosome monosomic addition line(CS-3C)were constructed and sequenced,and the key miRNAs and 24 nt siRNAs in Gc action were identified to investigate whether these two categories of small RNAs can participate in Gc action of wheat.The main research results are as follows:1.Identification and analysis of the key miRNAs:This study identified 239 conserved and 72 putative novel miRNAs.Among these 311 miRNAs,104 conserved and 31 putative novel miRNAs were differentially expressed between these two plant materials,of which 74 miRNAs were up-regulated in CS-3C,and 61 were down-regulated.The results of target genes prediction of differentially expressed miRNAs and GO analysis of the targets revealed that the functions of many target genes of these differentially expressed miRNAs maybe relevant to the Gc action.2.Verification of the differentially expressed miRNAs and their target genes:To verify the accuracy of the sequencing analysis results,4 differentially expressed conserved miRNAs were randomly selected to perform the TaqMan qRT-PCR.The high degree of consistency between sRNA sequencing data and TaqMan qPCR data indicated that the result of high-throughput sequencing data is credible.The expression patterns of 6 target genes of the 4 miRNAs above were analyzed by qRT-PCR and the results indicated that the expression profiles of target genes were negative correlated with their corresponding miRNAs.Finally,5?RACE were performed to further verify the cleavage site of target gene.3.Genetic transformation and functional analysis of tae-miR9657b-3p in rice:Cloned the sequence which contains pre-tae-miR9657b-3p and obtained the tae-miR9657b-3p over-expressed rice plants.Three lines which exhibited high levels of miR9657b-3p expression were used for subsequent analysis.QRT-PCR result of the target gene TC492017 indicated that it was down-regulated in transgenic rice,that is,miR9657b-3p was negative correlate with its target gene.Compared with wild-type lines,the fertility and pollen germination rate of the transgenic rice was reduced,indicating that over-accumulation of tae-miR9657b-3p can reduce plant fertility.Six fertility-related genes SPX1,GEN-L,UDT1,PAIR1,PAIR2 and REC8 were down-regulated in transgenic rice lines,which suggested that over-accumulation of miR9657b-3p affected the expression of these fertility-related genes,and further caused the decline in the fertility of transgenic rice.These results above indicated that tae-miR9657b-3p can participate in the regulation of plant fertility and may play an important role in the gametocidal phenomenon in wheat.4.Identification and differentially expressed analysis of 24 nt siRNAsAnalyzed the differences in distribution and density of 24 nt siRNAs on the corresponding chromosomes between CS-3C and CS wheat lines.The results of annotation analysis indicated that most of the siRNAs in the differential sliding windows corresponded to repeat sequences of the chromosomes,including some known types of TEs.Compared with CS,the expression level of 24 nt siRNAs was general lower in CS-3C.The distribution of siRNAs within the differential sliding windows in protein-coding genes,including both transcribed and 3 kb upstream and downstream flanking regions was examined.The results indicated that the highest density of 24 nt siRNAs occurred within 1 kb upstream and downstream of the transcribed regions in both CS and CS-3C.The result of the gene ontology(GO)analysis which was performed on genes associated with differentially expressed siRNAs within the transcribed and 1 kb upstream regions showed that these genes were enriched for the molecular functions “catalytic” and “binding” activities.5.Bisulfite sequencing analysis of 24 nt siRNA-associated transposonThe CHH methylation levels of four LINE/L1 retrotransposon were analyzed by bisulfite sequencing.The results indicated that CHH methylation levels were reduced in these TEs in CS-3C wheat line compared with those in CS.The differential accumulation of repeat-associated 24 nt siRNAs maybe associated with differential CHH methylation at the some of the TE sequences.The decrease of repeat-associated 24 nt siRNAs and the associated reduction in CHH methylation in the CS-3C line may cause genome instability and hence contribute to Gc action in wheat.