Identification of Novel and Conserved miRNAs in Leaves of In vitro Grown Citrus reticulata “Lugan” Plantlets by Solexa Sequencing

MicroRNAs (miRNAs) play essential roles in plant development, but the roles in the in vitro plant development are unknown. Leaves of ponkan plantlets derived from mature embryos at in vitro culture conditions were used to sequence small RNA fraction via Solexa sequencing, and the miRNAs expression was analyzed. The results showed that there were 3,065,625 unique sequences in ponkan, of which 0.79% were miRNAs. The RNA sequences with lengths of 18–25 nt derived from the library were analyzed, leading to the identification of 224 known miRNAs, of which the most abundant were miR157, miR156, and miR166. Three hundred and fifty-eight novel miRNA candidates were also identified, and the number of reads of ponkan novel miRNAs varied from 5 to 168,273. The expression of the most known miRNAs obtained was at low levels, which varied from 5 to 4,946,356. To better understand the role of miRNAs during the preservation of ponkan in vitro plantlet, the expression patterns of cre-miR156a/159b/160a/166a/167a/168a/171/398b were validated by quantitative real-time PCR (qPCR). The results showed that not only the development-associated miRNAs, e.g., cre-miR156/159/166/396, expressed highly at the early preservation period in the in vitro ponkan plantlet leaves but also the stress-related miRNAs, e.g., cre-miR171 and cre-miR398b, expressed highly at the same time. The expression levels of most tested miRNAs were found to decrease after 6 months and the amounts of these miRNAs were kept at low levels at 18 months. After analyzing the expression level of their targets during the reservation of the ponkan in vitro plantlet, development-associated cre-ARF6 and stress-related cre-CSD modules exhibited negative correlation with miR167 and miR398, respectively, indicating an involvement of the miRNAs in the in vitro development of ponkan and function in the conservation of ponkan germplasm.

[1]  Michelle T. Juarez,et al.  microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity , 2004, Nature.

[2]  Meng Wang,et al.  PsRobot: a web-based plant small RNA meta-analysis toolbox , 2012, Nucleic Acids Res..

[3]  Yijun Qi,et al.  DNA methylation mediated by a microRNA pathway. , 2010, Molecular cell.

[4]  V. Moulton,et al.  Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. , 2008, Genome research.

[5]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[6]  Xuemei Chen,et al.  microRNA biogenesis and function in plants , 2005, FEBS letters.

[7]  Nils Blüthgen,et al.  Targetfinder.org: a resource for systematic discovery of transcription factor target genes , 2010, Nucleic Acids Res..

[8]  Robert D. Finn,et al.  Rfam 12.0: updates to the RNA families database , 2014, Nucleic Acids Res..

[9]  D. Bartel,et al.  Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. , 2004, Molecular cell.

[10]  Hideki Makishima,et al.  [Deep sequencing]. , 2013, [Rinsho ketsueki] The Japanese journal of clinical hematology.

[11]  Tai Wang,et al.  Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa , 2011, Genome Biology.

[12]  R. Sunkar,et al.  Posttranscriptional Induction of Two Cu/Zn Superoxide Dismutase Genes in Arabidopsis Is Mediated by Downregulation of miR398 and Important for Oxidative Stress Tolerance[W] , 2006, The Plant Cell Online.

[13]  S. Hake,et al.  Big impacts by small RNAs in plant development. , 2009, Current opinion in plant biology.

[14]  C. Llave,et al.  High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.) , 2011, PloS one.

[15]  Seon-Ju Shin,et al.  miR171 Family Members are Involved in Drought Response in Solanum tuberosum , 2011, Journal of Plant Biology.

[16]  E. Benková,et al.  The Transcription Factors BEL1 and SPL Are Required for Cytokinin and Auxin Signaling During Ovule Development in Arabidopsis[W] , 2012, Plant Cell.

[17]  Chen Wang,et al.  Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata) , 2010, BMC Genomics.

[18]  R. Sunkar,et al.  Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis , 2009, Planta.

[19]  Xiu-Jie Wang,et al.  Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids , 2009, Proceedings of the National Academy of Sciences.

[20]  N. Chua,et al.  The role of miR156/SPLs modules in Arabidopsis lateral root development. , 2015, The Plant journal : for cell and molecular biology.

[21]  N. Ye,et al.  Identification of miRNA from Porphyra yezoensis by High-Throughput Sequencing and Bioinformatics Analysis , 2010, PloS one.

[22]  Z. Lai,et al.  Comparative Analysis Reveals Dynamic Changes in miRNAs and Their Targets and Expression during Somatic Embryogenesis in Longan (Dimocarpus longan Lour.) , 2013, PloS one.

[23]  Qian Luo,et al.  MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis. , 2010, Molecular plant.

[24]  D. Bartel,et al.  Criteria for Annotation of Plant MicroRNAs , 2008, The Plant Cell Online.

[25]  W. Karłowski,et al.  Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses , 2015, Front. Plant Sci..

[26]  V. Moulton,et al.  High-throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families , 2008, BMC Genomics.

[27]  Yingyin Yao,et al.  Cloning and characterization of microRNAs from wheat (Triticum aestivum L.) , 2007, Genome Biology.

[28]  Wen-Hsiung Li,et al.  Uncovering Small RNA-Mediated Responses to Phosphate Deficiency in Arabidopsis by Deep Sequencing1[W][OA] , 2009, Plant Physiology.

[29]  Heinz Saedler,et al.  The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis , 2008, Plant Molecular Biology.

[30]  Qiang Xu,et al.  Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange , 2011, Planta.

[31]  Wen-Wu Guo,et al.  Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type , 2010, BMC Genomics.

[32]  H. Liu,et al.  Development-associated microRNAs in grains of wheat (Triticum aestivumL.) , 2013, BMC Plant Biology.

[33]  Z. Yin,et al.  Identification and characterization of conserved microRNAs and their target genes in wheat (Triticum aestivum). , 2010, Genetics and molecular research : GMR.

[34]  K. Chong,et al.  Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response , 2009, BMC Genomics.

[35]  Li Li,et al.  Genome-wide comparison of microRNAs and their targeted transcripts among leaf, flower and fruit of sweet orange , 2014, BMC Genomics.

[36]  Ryan D. Morin,et al.  Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. , 2008, Genome research.

[37]  D. Bartel,et al.  MicroRNAS and their regulatory roles in plants. , 2006, Annual review of plant biology.

[38]  Ruiqiang Li,et al.  SOAP: short oligonucleotide alignment program , 2008, Bioinform..

[39]  Rui Chen,et al.  Identification of microRNAs in wild soybean (Glycine soja). , 2009, Journal of integrative plant biology.

[40]  Naama Menda,et al.  Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato , 2007, Nature Genetics.

[41]  Ramanjulu Sunkar,et al.  In silico identification of conserved microRNAs in large number of diverse plant species , 2008, BMC Plant Biology.

[42]  Shuhong Zhao,et al.  Discovery of Porcine microRNAs in Multiple Tissues by a Solexa Deep Sequencing Approach , 2011, PloS one.

[43]  Claude W dePamphilis,et al.  Conservation and divergence of microRNAs in Populus , 2007, BMC Genomics.

[44]  Qiang Xu,et al.  Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis. , 2015, Plant biotechnology journal.

[45]  Guiling Sun.  MicroRNAs and their diverse functions in plants , 2011, Plant Molecular Biology.

[46]  J. Reed,et al.  Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction , 2006, Development.

[47]  S. Smita,et al.  Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis , 2015, Front. Plant Sci..

[48]  E. Miska,et al.  MicroRNA functions in animal development and human disease , 2005, Development.

[49]  Detlef Weigel,et al.  Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana[W][OA] , 2008, The Plant Cell Online.

[50]  Y. Kamiya,et al.  IAA-Ala Resistant3, an Evolutionarily Conserved Target of miR167, Mediates Arabidopsis Root Architecture Changes during High Osmotic Stress[W] , 2012, Plant Cell.

[51]  Hirokazu Tsukaya,et al.  Mechanism of leaf-shape determination. , 2006, Annual review of plant biology.

[52]  Huaihua Liu,et al.  Identification and characterization of maize microRNAs involved in the very early stage of seed germination , 2011, BMC Genomics.

[53]  Han Xia,et al.  Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.) , 2010, BMC Plant Biology.

[54]  Identification and functional analysis of novel and conserved microRNAs in tomato , 2014, Molecular Biology Reports.