Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton [small RNA]
ABSTRACT: Genetic male sterility (GMS) in cotton (Gossypium hirsutum) plays an important role in the utilization of hybrid vigor. However, the molecular mechanism of the GMS is still unclear. While numerous studies have demonstrated that microRNAs (miRNA) regulate flower and anther development, whether different small RNA regulations exist in GMS and its wild type is unclear. To investigate the global expression and complexity of small RNAs during cotton anther development, three small RNA libraries were constructed from the anthers of three development stages each from fertile wild type (WT) and its GMS mutant cotton. Examination of different miRNA profiles in 2 lines.
Project description:In this study, in order to identify miRNA targets, a degradome library derived from anthers of the WT and GMS (Genetic Male Sterility) mutant representing three stages of development was constructed and sequenced, resulting in the generation of 24.6 million raw reads. After removal of low quality sequences and adapter sequences, 24.4 million clean reads were obtained and 98% were 20 or 21 nt in length as expected in that normally length distribution peak of degradome fragment is between 20 and 21 nt [Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ: Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 2008, 18:758-762]. Identification of miRNA targets in the WT and GMS muant anthers. Anthers of the WT and GMS mutant representing three stages of development [the meiosis stage (WT: Mar-F-1; mutant: Mar-S-1) and tetrad stage (WT: Mar-F-2; mutant: Mar-S-2), together with the uninucleate microspore stage (WT: Mar-F-3; mutant: Mar-S-3) from the GMS ‘Dong A’ mutant and its fertile wild type] were collected during early mornings.
Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are short (19–25 nucleotides) non-coding RNA molecules that have large-scale regulatory effects on development and on stress responses in plants.The objective of this study is to investigate the transcriptional profile of miRNAs and other small non-coding RNAs in Verticillium–inoculated cotton roots. Four small RNA libraries were constructed from mocked and infected roots of two cotton cultured species which are with different Verticillium tolerance (‘Hai-7124’, Gossypium barbadense L., a Verticillium-tolerant cultivar, and ‘Yi-11’, Gossypium hirsutum L. a Verticillium-sensitive cultivar). The length distribution of obtained small RNA pools was significantly different among libraries. A total of 215 conserved miRNA families were identified in the two cotton species, of them 14 are novel. There were >65 families with different expression between two libraries. We also identified two ta-siRNAs and thousands of endogenous siRNA candidates, and hundred of them exhibited altered expression after inoculation of Verticillium. The profiling of these miRNAs and other small non-coding RNAs lay the foundation for further understanding of small RNAs function in the regulation of Verticillium defence responses in cotton roots. Examination of 2 different traetments in 2 cotton types.
Project description:We profiled and quantitated miRNAs in two skin tumors (Basal cell carcinoma and Merkel cell carcinoma) and identified tumor-specific miRNAs. We used these tumor-specific miRNAs to guide development of miRNA fluorescence in situ hybridization. 2 barcoded sequencing runs, including 40 unique samples (36 used in manuscript). The details of each sample can be found in Supplementary Tables S1 and S2.
Project description:To identify potential miRNAs involved in fiber development and elucidate their expression differences between G. barbadense and G. hirsutum, we constructed two small RNA libraries, Gb10 and Gh10, prepared from fibers of 3-79 (G. barbadense) and TM-1 (G. hirsutum) collected at 10 days post-anthesis (DPA). We identified 28 conserved miRNA families, including 24 that exactly match known plant miRNA families in miRBase. With MIREAP and newly developed software miRsearcher, 7 candidate-novel miRNAs were found. 5 candidate-novel miRNAs were expressed in both species, 2 candidate-novel miRNAs were expressed only in one species. Moreover, 4 miRNA families showed significant expression differences between sea-island cotton and upland cotton in 10 DPA fibers. two examples including 3-79 and TM-1 10 DPA fibers
Project description:MicroRNAs (miRNAs) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 non-invasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed-sequence-dependent miRNA-target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most non-invasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including mir-98/let-7), with most miRNA changes apparent already in the non-invasive carcinomas. In addition, patients that went on to develop metastasis demonstrated increased expression of mir-423, and triple negative breast carcinomas were most distinct from other tumor subtypes due to up-regulation of the mir-17~92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible, but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested. 21 barcoded sequencing runs, including 185 unique samples and 54 samples in replicate (6 in triplicate and the remaining in duplicate). The details of each sample can be found in Supplementary Tables S1 and S2.
Project description:The degradation and 3′ end modification of plant microRNAs (miRNAs) play crucial roles in regulating miRNA function and stability. However, the process and mechanism of miRNA degradation and 3′ end modification has, to date, been poorly characterized. Here, we report that analysis of the two small RNA libraries constructed from two hickory floral differentiation stages by deep sequencing obtained a large number of truncated miRNAs and miRNAs with 3′ end modifications. The presence of so many truncated miRNAs suggests that plant miRNAs may be degraded through the 5′ to 3′ and 3′ to 5′ ends simultaneously, but the probability of miRNAs being truncated from the 3′ end was higher than from the 5′ end. Single- or double-nucleotide 3′ additions to miRNAs has been observed in many families. In this study, the 3′ addition of adenine to miRNA was the most common, accounting for more than 50% of all miRNA 3′ end modification in both small RNA libraries, followed by uridine addition. This suggests that the 3′ end modification of miRNAs shows a bias towards adenine and uridine in plants. Furthermore, we observed that both truncated miRNA and isomiR expressions associated with mature miRNAs. Our study provides more information regarding the degradation and 3′ end modification of miRNAs in plants. Examination of 2 different female flower buds
Project description:Background: Dwarf cottons are more resistant to damage from wind and rain and associated with stable, increased yields, and also desirable source for breeding the machine harvest varieties. In an effort to uncover the transcripts and miRNA networks involved in plant height, the transcriptome and small RNA sequencing were performed based on dwarf mutant Ari1327 (A1), tall-culm mutant Ari3697 (A3) and wild type Ari971 (A9) in Gossypium hirsutum. Results: The transcriptome sequencing analysis showed that the enriched pathways of top 3 differentially expressed genes (DEGs) were categorized as carotenoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction in both A1-A9 and A3-A9. The ABA and IAA related factors were differentially expressed in the mutants. Importantly, we found the lower expressed SAUR and elevated expressed GH3, and ABA related genes such as NCED and PP2C maybe relate to reduced growth of the plant height in Ari1327 which is consistent with the higher auxin and ABA content in this mutant. Furthermore, miRNA160 targeted to the auxin response factor (ARF) and miRNA166 (gma-miR166u and gma-miR166h-3p) targeted to ABA responsive element binding factor were related to the mutation in cotton. We have noticed that the cell growth related factors (smg7 targeted by gra-miR482 and 6 novel miRNAs and Pectatelyases targeted by osa-miR159f), the redox reactions related factors (Cytochrome P450 targeted by miR172) and MYB genes targeted by miR828, miR858 and miR159 were also involved in plant height of the cotton mutants. A total of 226 conserved miRNAs representing 32 known miRNA families were obtained, and 38 novel miRNAs corresponding to 23 unique RNA sequences were identified. Total 531 targets for 211 conserved miRNAs were obtained. Using PAREsnip, 27 and 29 miRNA/target conserved interactions were validated in A1-A9 and A3-A9, respectively. Furthermore, miRNA160, miRNA858 and miRNA172 were validated to be up-regulated in A1-A9 but down-regulated in A3-A9, whereas miRNA159 showed the opposite regulation. Conclusions: This comprehensive interaction of the transcriptome and miRNA at tall-culmand dwarf mutant led to the discovery of regulatory mechanisms in plant height. It also provides the basis for in depth analyses of dwarf mutant genes for further breeding of dwarf cotton. Total RNA was purified from stem apexes of three samples at the fifth true leaf stages and sequenced deeply using Illumina HiSeq 2000 system.
Project description:Clonorchis sinensis is a zoonotic parasite causing clonorchiasis associated with human diseases such as biliary calculi, cholecystitis, liver cirrhosis, and is classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules essential for the complex life cycle of parasites and involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing, bioinformatic predictions with stem-loop real-time PCR analysis. Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belong to 284 families. There is strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil is the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and the end of conserved miRNAs. There is no significant “seed region” at the first and ninth positions commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis are still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There are two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping a stringent conserved seed region with high active innovation in other place of miRNA mainly in the middle and the end, which are perfect for the parasite to perform its complex life style and for host changes. The present study represents the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as the miRNA studies of other related species such as Opisthorchis felineus and O. viverrini of human and animal health significance. Analysis of miRNA profile in parasite of C. sinensis
Project description:In plants, MicroRNAs (miRNAs) are a new class of endogenous small RNAs that play essential regulatory roles in plant growth, development and stress response. Extensive studies of miRNAs have been performed in model plants such as rice, Arabidopsis thaliana and other plants. However, the number of miRNAs discovered in maize is relatively low and little is known about miRNAs involved in the four stages during maize ear development. Here, we use deep-sequencing, miRNA microarray assays and computational methods to identify, proﬁle, and describe conserved and non-conserved miRNAs at four developmental stages. A total of 27 conserved miRNA families were identiﬁed in all four stages, In addition to known miRNAs, we also found 23 new maize-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. We have found that many known and new miRNAs showed temporally expression. Finally, a total of 251 transcripts (140 genes) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.This study led to the confirmation of the authenticity of 27 conserved miRNA families and the discovery of 23 novel miRNAs in maize. In addition, we have identified 130 targets of known and new miRNAs and ta-siRNA using recently developed tools for the global identification of miRNA targets. Identification and characterization of this important class of regulatory genes in maize may improve our understanding of molecular mechanism controlling flower development. The seeds of maize inbred line B73 were first sterilized and germinated in an incubator, then grown in a controlled environment with 28°C/21°C (day/night) under a 16-h day/8-h night photoperiod with a relative humidity of 70%. Ear development can be divided into four stages: the growth point elongation phase, spikelet differentiation phase, the floret primordium differentiation phase and floret organ differentiation phase. Plant materials (ears) were collected as described previously. In brief, ears were manually collected at the four developmental stages according to the plant features (number of visible leaves, leaf age index, and number of unfolded and folded leaves) combined with microscopic observation.