Comparative Analyses Of Non-coding Rnas In Aurantioideae And The Functional Study Of Csi-miR164a

As the two main types of non-coding RNAs(nc RNAs),long non-coding RNAs(lnc RNAs)and micro RNAs(mi RNAs)play important roles in various biological processes in plants.Lnc RNAs and mi RNAs have been identified in many plants.However,little is known about the evolutionary characteristics of lnc RNAs or mi RNAs among closely related plant species.Citrus is one of the most important fruit crops containing abundant species resources and genome resources,such as sweet orange(Citrus sinensis),pummelo(Citrus grandis)and clementine(Citrus clementina).It is also of great value to explore lnc RNAs and mi RNAs that involed in citrus fruit development.Here,we present a large-scale comparative study of linc RNA(long intergenic noncoding RNA)and mi RNA transcription patterns among Aurantioideae species,respectively.We revealed the conservation of linc RNA transcription in nine citrus species,and demonstrated a mi R166-linc RNAs regulatory network that may be involed in fruit development.Comparative analyses of mi RNAs among Aurantioideae species elucidated the conservetion of mi RNAs.We also identified a set of fruit-preferential expressed mi RNAs containing mi R164,which is preferentially and highest expressed in fruits of multiple Aurantioideae species.Functional study of csi-mi R164 a revealed that mi R164 have impacts on the morphology and senescence of citrus organs.Detail results are showed as follows:1.Conservation of linc RNA transcription among Aurantioideae species.By strand-specific RNA-sequencing,we identified 18075 linc RNAs(14575 linc RNA loci)from 34 tissue samples of nine citrus species.The results indicated that the evolution of linc RNA transcription is more rapid than that of m RNAs.In total,82.8%–97.6% of sweet orange linc RNA genes were shown to have homologous sequences in other citrus genomes.However,only 15.5%–28.8% of these genes had transcribed homologous linc RNAs in these citrus species,presenting a strong contrast to the high conservation of m RNA transcription(81.6%–84.7%).Evolutionarily conserved linc RNAs showed higher expression level and lower tissue specificity than species-specific linc RNAs.Notably,we observed a similar tissue expression pattern of homologous linc RNAs in sweet orange and pummelo,suggesting that these linc RNAs may be functionally conserved and selectively maintained.We also identified and validated a linc RNA with the highest expression in fruit that acts as an endogenous target mimic(e TM)of csi-mi R166 c,and two linc RNAs that act as a precursor and target of csi-mi R166 c,respectively.These linc RNAs together with csimi R166 c could form an e TM166-mi R166c-targeted linc RNA regulatory network that possibly affects citrus fruit development.2.Comparative analyses of mi RNAs among Aurantioideae species and identification of mi RNA candidates that involed in fruit development.By small RNA sequecing,we identified 285 unique mi RNAs(693 MIRNA genes)from15 tisssue samples of five Aurantioideae species.Among these mi RNAs,174 mi RNAs were from 45 mi RNA families with homologs in mi RBase,and therefore we labeled these as“known mi RNAs”.Conversely,the rest of 111 mi RNAs were called as “novel mi RNAs”.Comparative analyses among five species revealed that 57.5% of known mi RNAs were expressed in at least two species.However,for novel mi RNAs,85.6% of them were specifically expressed in one species.These results indicated that the transcriptional conservation of novel mi RNAs were lower than that of known mi RNAs.On the whole,the expression of most novel mi RNAs were at relatively low levels when compared with known mi RNAs.We also found that the expression pattern of 41 conserved mi RNAs in five species were highly conserved.We also identified 23 mi RNAs that were tissuepreferential expressed in at least two species,among them 13 mi RNAs were preferentially expressed in fruits,which were regarded as candidate mi RNAs that may be involed in fruit development.3.Function analyses of csi-mi R164 a in citrus.Analyses of small RNA sequencing data revealed that mi R164 were preferentially expressed in fruits of five Aurantioideae species.In order to study the function of mi R164,we overexpressed Csi MIR164 A gene under the Ca MV35 S promotor in early flowering mutant of trifoliate orange.Compared with wild type,the expression levels of mi R164 a and four mi R164a-targeted NAC transcription factors in three transgenic lines were notably incresed and decreased,respectively.Compared with wild type,transgenic lines displayed fused leaves,dislocation of buds and leaves,delayed falling of leaves,styles and stigmas.These results suggested the conserved function of csi-mi R164 a in organ boundary establishment and leaf senescence in plants and the novel role of mi R164 a in fruit style and stigma development in citrus.