Transcriptome Analysis And Regulatory Mechanism Of MiRNAs Involved In Embryogenic Callus Formation In Wheat(Triticum Aestivum L.)

The feasible and efficient tissue culture plays an important role in the plant genetic engineering.However,the efficiency of In vito culture is low as one of the main factors that limits transgenic technology in wheat(Triticum aestivum L.).Wheatimmature embryos(IMEs)are preferred for tissue culture to mature embryos(MEs)because IMEs are easily generate embryogenic calli,producing large number of plants.The molecular mechanisms of regulation and the biological pathways involved in embryogenic callus formation in wheat remain unclear.In this study,the culture condiation for immature embryos was optimized.Meanwhile,the difference of embryogenic callus development in immature and mature embryos was observed.Further,Deep-sequencing were employed to explore the molecular mechanism of embryogeinc callus formation for immature and mature embryos when cultured at 3DC、6DC and 15DC in transcriptome and miRNA.Differentially expressed genes with potentially important roles in embryogenic calli formation and miRNAs potentially involved in the embryogenic calli formation were analyzed.Results are as follows:1.The genetype Zhoumai 18 and Yunong 201 have high culture efficiency.As the age of immature embryos at 10 days after follower the embryos have higher embryonic callus induction efficiency,but have lower callus differentiation efficiency.At 18 days after flower,the callus differentiation efficiency and embryonic callus induction efficiency become lower,in total,the age of 12d to 16d of immature embryos are subtible for tissue culture,but the most suitable time for tissure culture is the age of 14 days after flower.2.The immature and mature embryos in the same genetype have different culture responce,such as zhoumai18,there obvious difference between them in embryogenic callus formation and callus dedifferentare,IMEs at 3 DC(IME3)main developed from scutellums(horizontal development),whereas ME3 main developed from germ layers at 6 DC,granular embryogenic calli were induced from IMEs(IME6),while the calli of ME6 enlarged sequentially and turned transparent.Somatic embryogenesis was visible in IMEs at 15 DC(IME15),while there was no embryogenic callus in ME15s.Therefore,embryogenic calli were obtained from IMEs but not from MEs at 15 DC.These results allowed us to study the embryogenic callus formation in transcriptome and miRNAs between the two types of calli at the formation stage.3.Calli from IMEs and MEs at 3 DC,6 DC,and 15 DC were used to construct six transcriptome libraries.All libraries were sequenced and 155,192,839 high quality paired-end reads were obtained from the six libraries.Our de novo assembly generated 9,247,796 contigs,331,201 transcripts with an average size of 1009.69 bp and 142,221 Unigenes with average length of 657 bp.Among these Unigeness,129,022(90.72%)were annotated with a significant Blastx hit.4.Comparison of calli transcriptomes showed that 3,181、2,085 and 1,468 uigenes were differentially expressed(P<0.05)between the two types of libraries at 3 DC,6 DC,and 15 DC,respectively.Between stages in IMEs and IMs libraries,1,536,1,683,and differentially expressed Unigenes were identified in IME6 vs.IME3,IME15 vs.IME6,ME6 vs.ME3 and ME15 vs.ME6 comparisons,respectively.Many TFs those were known involved in meristem activity and redifferentent shown differentally expressed in IMEs vs.ME comparisons,and in comparisons between stages in IME.Genes that involved in hormonal responses and stress were also differentially expressed.5.Calli from IMEs and MEs at 3 DC,6 DC,and 15 DC were used to construct six sRNA libraries.All libraries were sequenced and 16-24 million raw reads were produced from each library.After the removal of adapter sequences,sequences of less than 18 nt and more than 30 nt in length,and low quality reads,10.8 million(mean;range 7.5-13.1 million)clean sRNAs were obtained from each library,and 3.5 million(mean;range 1.5-6.3 million)of them were unique reads.A total of 85 known miRNAs were identified,of which 30,33,and 18 were differentially expressed(P<0.05)between the IME and ME libraries at 3 DC,6 DC,and 15 DC,respectively.6.Additionally,171 novel and 41 candidate miRNAs were also identified,of the novel miRNA,69,67,and 37 were differentially expressed(P<0.05)between the two types of libraries at 3 DC,6 DC,and 15 DC,respectively.The expression patterns of eight known and eight novel miRNAs were validated using quantitative real-time polymerase chain reaction.Gene ontology annotation of differentially expressed miRNA targets provided information regarding the underlying molecular functions,biological processes,and cellular components involved in embryogenic callus development.Functional miRNAs,such as miR156,miR164,andmiR1432,differentially expressed in IMEs and MEs might be related to embryogenic callus formation and somatic embryogenesis.